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Localizations of α1 and β1 subunits of soluble guanylate cyclase in the rat brain
Molecular Bram Research, 20 (1993) 335-344 © 1993 Elsewer Soence Pubhshers B V All rights reserved 0169-328x/93/$06 00
335
BRESM 70669
Localizations of a l and/31 subunits of soluble guanylate cyclase in the rat brain T
Furuyama,
S
Inagakl
and
H
Takag~
First Department of Anatomy, Osaka City Untt,erstty Medical School, Osaka (Japan) (Accepted 4 May 1993)
Key words Soluble guanylate cyclase, Soluble guanylate cyclase a l subunlt, Soluble guanylate cyclase/31 subumt, Rat brain In sltu hybridization
We studied the Iocahzatlons of a l and/31 subumts of soluble guanylate cyclase using m sltu hybridization The/3 subunlt was widely distributed m most neurons throughout the brain, with different levels of expression The ctl subumt was also dl~tnbuted throughout the brain, however, it was located in more hmlted regions Both subunlts were expressed markedly In the glomerular layer of the olfactory bulb, dorsal and ventral strlatum, and several regions in the bralnstem Regions with httle or no a l subumt expression, but with marked expression of the /31 subumt included the olfactory bulb except for the glomerular layer, pyramidal cell layer m CA1 and granular cell layer in the dentate gyrus of the h~ppocampus, and many bramstem nuclei The above regmns expressing both subumts are suggested to contain active soluble guanylate cyclase as a target for mtnc orade, and thus may be revolved m cellular signal transductlon
membrane
INTRODUCTION
a n d a c t i v a t i n g S G C to i n c r e a s e c G M P
fol-
lowing stimulation of excitatory ammo acid receptors Guanylate
cyclases
3',5'-monophosphate
(GCs)
form
cychc
guanoslne
m the bram 6
( c G M P ) , a n m t r a c e l l u l a r signal-
ing m o l e c u l e p r e s e n t in v a r i o u s tissues, i n c l u d i n g t h e b r a i n G C s exist m s o l u b l e a n d m e m b r a n e - b o u n d
S o l u b l e G C ( S G C ) ( E C 4 6 1 2) is r i c h m l u n g a n d b r a i n a n d is a c t w a t e d by n i t r i c o x i d e ( N O ) 26 18 S G C contains heme, a putatwe
MATERIALS AND METHODS
forms
r e c o g n i t i o n s~te f o r N O , a
s t i m u l a t o r for this e n z y m e 6'~8. S G C p u r i f i e d f r o m l u n g h a s a m o l e c u l a r m a s s o f a b o u t 150 k D a a n d exists as a
Animals Five W~star rats weighing 150 g were used Rats were decapitated under sodium pentobarbltal anesthesia (50 mg/kg, i p ) The brams were qmckly removed and frozen in powdered dry ice Serial sectmns m 15/~m thickness were cut on a cryostat, thaw-mounted on sdyated glass slides and kept at -70°C untd they were ready for use for m sltu hybridization
h e t e r o d l m e r o f 82 k D a ( a ) a n d 70 k D a (/3) s u b u m t s 9 Recently, cDNA for each subumt has been cloned f r o m b o v i n e a n d r a t l u n g ~°'t1,~3,a5 E x p r e s s i o n o f o n l y o n e o f t h e s e s u b u m t s in m a m m a h a n cells r e s u l t e d m n o d e t e c t a b l e G C a c t w l t y 13'~5 C o e x p r e s s l o n o f b o t h a a n d / 3 s u b u n l t s a p p e a r s to b e n e c e s s a r y f o r S G C actlvltys ~s A l t h o u g h t h e l o c a h z a t l o n o f t h e /31 s u b u n l t m t h e b r a i n has b e e n
s t u d l e d by m sltu h y b r i d i z a t i o n ,
there are no reports concerning the locahzat~on of the a
subunlt
In the
present
study,
we
examined
the
d i s t r i b u t i o n o f t h e a l a n d /31 s u b u n l t s o f S G C m t h e rat b r a i n W e also a t t e m p t e d to clarify t h e t a r g e t s o f N O , a g a s e o u s m e s s e n g e r c a p a b l e o f c r o s s i n g t h e cell
In sltu hybndtzanon In SltU hybridization was carried out using methods described previously 16 After drying the shde-mounted sections at room temperature, the tissue sections were fixed m 4% paraformaldehyde m 0 1 M phosphate buffer (pH 7 2) for 10 mm, rinsed with phosphatebuffered saline (PBS) three times, and dehydrated in a graded series ot ethanols and chloroform The hybridization was performed overmght at 42°C m a buffer containing 4×SSC, 50% formamlde, 0 12 M phosphate buffer, l×Denhardt's solution, 02% sodmm dodecylsulfate, 250 ~ g / m l yeast RNA, 10% dextran sulfate, 100 mM dlthlothreltol with 106 cpm of labeled probe m 150 p.l buffer per shde Following hybridization, the sections were rinsed m I × S S C three times each for 20 mln at 55°C, this was followed by brief dehydration with 70% and 100% ethanol The tissue sections were then exposed to X-ray film (Kodak, XAR or Amersham, Hyperfilm /3-max) for 1 week to obtain for film autoradlograms, or were coated
Correspondence T Furuyama, First Department of Anatomy, Osaka City Umverslty Medical School, 1-4-54 Asahlmachl, Abeno-ku, Osaka, 545 Japan Fax (81)6-646-6611
336 with lltord K-5 emulMon (diluted 1 1 wtth distilled water at 40°C) and exposed for 4 - 6 weeks at 4°C in light-tight boxes for mlcroautoradiography Following development in D19 (Kodak) the sections were counterstamed with thionm and then observed using dark- and bright-field microscopes (Olympus) Neurons with grain densities at least 3 times higher than the background density were considered positively labelled As a control a competition experiment was done The brain sections were hybridized with labeled ohgonucleottde probes together with 100X
1
excess unlabeled probe No s p e o h c s~gnaN were detected alter the c o m p e t m o n experiment
Ohgonut leottde probe~
Ohgonucleotlde probes were synthesized in an Apphed Blosysterns D N A synthesizer and purified using a N E N S O R B P R E P column (NEN) The a l and /31 subumt probes were complementary to bases 718-766 of the rat SGC (~1 s u b u m t I~ and 575-623 of the rat
al
2
,81 A
I
B
r k UI
r k
v
r ¸
E
r
k
SN
Figs 1 and 2 Fdm autoradlograms showing the dmtnbutlon ol the SGC a l s u b u m t (left side, Fig 1) and /~1 subumt (right side Fig 2) Frontal sections of the rat brain are arranged from rostral to caudal, A - K 3 oculomotor nucleus, 7 facial nucleus, A, accumbens nucleus, AP area postrema, CG cmgulate cortex, CP, caudate putamen, D R , dorsal raphe nucleus LC, locus coeruleus, LD, laterodorsal tegmental nucleus M H medial habenular nucleus OT, olfactory tubercle, P, piriform cortex PB, parabigemInal nucleus, PG pineal gland, R red nucleus RT, reticular thalamlc nucleus, S, subiculum, SC, superior colhculus SN substantla mgra V vestibular nuclei
337 /31 subumt ~5, respectively These ollgonucleotldes were 3'-endlabeled with terminal deoxynucleotldyl transferase (Takara Shuzo) and [a-35S]dATP (NEN) to a specific actwlty of 109 cpm//xg Nomenclature used followed that m the Paxlnos and Watson atlas 17
RESULTS T h e a l s u b u n l t of S G C was p r e s e n t t h r o u g h o u t the b r a i n b u t ~ts expression was h m l t e d to several regions
B1
ctl 1
I:
2
G
H
K
Figs 1 and 2 (continued)
338 (Fig 1), while the /31 subunit of SGC was more widely distributed throughout the brain with differences in the level of the expression (Fig 2) SGC was usually present in neuronal cells (Fig 3) Both of the subunits were expressed abundantly in several forebraln and brainstem regions Furthermore, the /31 subunlt was expressed in many nuclei of the forebraln and brainstem in addition to the regions described above, m which both subunlts were abundant In some regions, such as the area postrema and pineal gland, the expression of the /31 subunlt was very weak, but that of the a l subunlt was strong Table I summarizes the intensities of expression in various regions
strongest expression of the a l subunlt in the rat brain (Fig 1 A - D ) The /31 subunit was also expressed strongly in these regions (Fig 2 A - D ) Numerous neurons expressed strong signals for both subunlt in the caudate putamen and fundus strlatl (Fig 4C,D) The globus palhdus and entopeduncular nucleus exhibited weak to moderate expression for both subunlts on fllm-autoradlograms (Fig 1A-C), while on mlcroautoradlograms there were scattered neurons but each neuron showed moderate to strong expression for both signals (Fig 4C,D) In the cerebral neocortex, the expression of the a l subunit was strong in the surface layer, layers II and III, weak In the deep layer, layers V and VI, and very weak In layer IV (Fig 1) The/31 subunlt was strongly expressed throughout the cortex, with the strongest expression seen in layer V and weak expression in layer IV (Fig 2) Expression of both a l and/31 subunlts was strong in the plrlform cortex and clngulate cortex (Figs l, 2 and 4A) In the clngulate cortex, a 1 expression was strongest in the surface layer at rostral level (Fig 1A,B), while/31 expression was strong in both surface and deep layers (Fig 2A,B) On film-autoradlograms, the density of the /31 subunit was higher than that of the a l subunlt in the hlppocampus (Figs 1D,E and 2D,E) The a l subunlt was strongly expressed in scattered, non-pyramidal and non-granular cells of the hlppocampus, moderately expressed in pyramidal cells of CA2 and CA3, and weakly expressed in pyramidal cells of CA1 and granular cells of the dentate gyrus (Fig 5A) There was also moderate a l subunit expression m the sublculum On the other hand, the /31 subunlt strongly expressed in both pyramidal and non-pyramidal cells of C A 1 - C A 3 , granular and non-granular cells of the dentate gyrus, and in the sublculum (Fig 5B) In the amygdala, expression ot the a l subunlt was
Forebram In the olfactory bulb, the a l subunIt was strongly expressed in the glomerular layer but weakly expressed m the other layers, whereas the /31 subunlt was expressed abundantly In many neurons of most layers of the olfactory bulb including the glomerular, mitral cell and internal granular cell layers In the anterior olfactory nuclei, the expression of the a l subunlt was moderate to strong, and that of the/31 subunlt was strong Neither subunlt was detected in the ependymal and subependymal layers The expression of the a l subunlt was very strong in the olfactory tubercle and accumbens nucleus (Figs 1A, 3 and 4A), strong in the tenla tecta and bed nucleus of the stria termlnahs, and weak to moderate in the septal nuclei and nuclei of the diagonal band Positive neurons were scattered in the substantla lnnomlnata The /31 subunIt was strongly expressed m the regions with moderate to strong expression of the a l subunlt and in the regions such as the islands of Calleja, where the a l subunit was weakly expressed (Figs 2A,B and 4B) The caudate putamen and fundus strlatl showed the
|
)
•
al
i
P
)O Fig 3 Bright-field photomicrographs of the accumbens nucleus Signals for the a l (A) and /31 (B) subumts are present over neuronal cell bodies Bar = 10 p.rn
339 TABLE I
Summary of the mRNA expression of the oil and/31 subuntts of SGC in the rat brain by In sttu hybridization + + + +, very strong, + + +, strong, + +, moderate, +, weak, +, very weak (arbltary umts) al
/31
+ + + + + + +
+ + + +
+ + + +
+ + + +
+ + + + + + + + + + +
+ + + + + + + + + + +
+ + + + + + + + + + +
+ + +
+ + + + + + ___ + +
+ + + + +
+ +
+ + + + + +
+ + + + + +
Hippocampus p y r a m i d a l cells in t h e C A 1 p y r a m i d a l cells in t h e C A 2 p y r a m i d a l cells In t h e C A 3 n o n - p y r a m i d a l cells dentate gyrus sub~culum
+ + + + + +
+ + + + + +
Amygdala lateral nucleus basolateral nucleus intercalated nucleus central nucleus
+ + + + + + +
+ + + + + + +
Thalamus medml habenula reticular thalamlc nucleus ventromedlal thalamlc nucleus zona mcerta parafasc~cular nucleus posterior mtralammar thalamlc nucleus
+ + + + + +
+ + + + + +
+ + + + + + + + + +
Hypothalamus paraventrlcular nucleus magnocellular subdwlslon p a r v o c e l l u l a r subdwls~on supraopt~c nucleus lateral hypothalamlc nucleus
+ + + + + + + +
+ + + +
+ + + + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
Forebram Olfactory bulb glomerular layer m l t r a l cell l a y e r internal granular layer anterior olfactory nucleus tema tecta accumbens nucleus septal nuclei nuclei of the diagonal band olfactory tubercle islands Calleja bed nucleus of the stria termlnahs caudate putamen f u n d u s strmtl globus palhdus entopeduncular nucleus Neocortex l a y e r II l a y e r III layer IV layer V layer VI p~rlform c o r t e x cmgulate cortex
+ + + + +
+ + + + + + + + + + + + +
+ + + + +
+ + + + + + +
+ + +
+ + + + + +
+ + + + + +
Dtencephalon + + + + + + + + + +
Mtdbram substantla mgra pars compacta substantla mgra pars retlculata lnterpeduncular nucleus Darkschewltsch nucleus r e t r o r u b r a l field a n d n u c l e u s raphe nuclei central grey oculomotor nucleus parab~gemmal nucleus
+ + + + + + + + + +
+ +
+ + +
34O TABLE I (continued) c~l
/31
+ + +
I-++ ++
+++
+++
+ ++++ ++++
+++ +++ +++ +++
Mtdbratn
superior colhculus superficial gray layer other layers inferior colhculus
++
Pons and medulla oblongata
pedunculopontme nucleus pontme nucleus laterodorsal tegmental nucleus locus coeruleus lateral parabrachial nucleus dorsal tegmental nucleus raphe nuclei reticular formation cochlear nucle~ vestxbular nuclm racial nucleus abducens nucleus nuclei of the tngemmal complex prepositus hypoglossalnucleus vagal complex amb~guus nucleus hypoglossalnucleus inferior olive area postrema
++ ++ + ++
+++ +++ +++ +++
+ +4
+++ +-++
+ ++
+++ +++
+ ++
+++ +++ +++ +++
++ ++ +
+++
+ +++
+
Cerebellum
Purkmje cells molecular layer granular cell layer cerebellar nuclm
++
++
+ +
+ + +++
Other regions
pineal gland
++++
moderate in the lateral, basolateral, and intercalated amygdaloid nuclei, weak in other amygdalold nuclei, including the basomedlal, medial, anterior and posterior amygdalold nuclei, and very weak in the central amygdalold nucleus /31 Subunlt expression was rather even, being moderate, throughout the amygdala, except in the central amygdaloid nucleus, where the expression was very weak Dtencephalon
Expression of the a l subumt was detected in several limited regions The medial habenula was one of the regions of the brain showing the strongest expression of both a l and 131 subunlts (Figs 1D and 2D) Moderate to strong expression of c~l and /31 subunlts was also noted In the reticular and ventromedial thalamlc nuclei, and in the zona lncerta (Figs 1C,D and 2C,D) The expression of the a l subunit was moderate in the parafasclcular nucleus and posterior lntralamlnar thalamlc nucleus, and weak or very weak in other regions of the thalamus On the other hand, the /31 subunit was distributed more evenly with moderate to strong expression throughout the thalamus, with the expres-
sion being stronger in the reticular and ventromedial thalamlc nuclei, and relatively weak in the ventral posterolateral and ventral posteromedlal thalamic nuclei The pretectal nuclei showed weak to moderate expression of the a l subunlt and moderate to strong expression of the/31 subunlt In the hypothalamus, a l subunlt was strongly expressed in the magnocellular subdivision of the paraventrlcular hypothalamlc (Fig 5C) and supraoptic nuclei, whereas it was weakly expressed in other hypothaiamlc nuclei In contrast, the/31 subunlt expression was more even, and moderate in most hypothalamlc nuclei, with relatively stronger expression m the magnocellular and parvoceUular subdivisions of the paraventricular nucleus and in the lateral hypothalamlc nucleus Mtdbram
Expression of the a l subunit was strong in the oculomotor nucleus, parablgemlnal nucleus (Fig 1G), and raphe nuclei including the dorsal raphe nucleus (Fig IG), moderate in the interpeduncular and Darkschewitsch nuclei, central gray and retrorubral field and nucleus, and weak in other mldbraln regions in-
341 c l u d l n g t h e s u p e r i o r a n d i n f e r i o r c o l h c u h ( F i g 1) S c a t t e r e d n e u r o n s m t h e d e e p m e s e n c e p h a h c nucleus w e r e m o d e r a t e l y positive for t h e a l s u b u n t t B e t a 1 s u b u n i t e x p r e s s i o n was m o r e even, b e m g m o d e r a t e to s t r o n g e x p r e s s i o n t h r o u g h o u t t h e m l d b r a l n , with relattvely s t r o n g e x p r e s s i o n m t h e s u b s t a n t l a m g r a p a r s c o m p a c t a a n d r e t l c u l a t a (Figs 2E a n d 5 D ) a n d the b o t t o m layer o f t h e superficial gray l a y e r o f t h e s u p e -
al
r l o r colhculus ( F i g 2 E - G ) , in a d d i t i o n to the nuclei exhibiting m o d e r a t e to s t r o n g a l e x p r e s s i o n
Pons and medulla oblongata T h e e x p r e s s i o n o f the /31 r a t h e r even, b e i n g m o d e r a t e to pons and medulla oblongata, s u b u n t t was h e t e r o g e n e o u s a n d
s u b u m t was g e n e r a l l y strong, t h r o u g h o u t the whtle t h a t of the a l h m i t e d to several nu-
.6'1
Fig 4 Dark-field photomicrographs showlrig the a l (A,C,E) and /31 (B,D,F) subumts m regions In which both subunlts are strongly expressed Frontal sections through the olfactory tubercle (A,B), caudate putamen (C,D) and locus coeruleus (E,F) Strong or very strong expression of the a l and/31 subunlts Is seen m the olfactory tubercle (OT) and preform cortex (P), caudate putamen (CP) and locus coeruleus (LC), and moderate expression is observed m scattered cells in the globus palhdus (GP), reticular thalamlc nucleus (RT) and raphe pontls (RP) Bars m A - E = 100 g.m, m F = 100 p.m
342 clear groups The laterodorsal tegmental nucleus and locus coeruleus very strongly expressed both a l and/31 subunlts (Figs 1H,I, 2H,I and 4E,F) In addition, the expression of the a l subunlt was moderate to strong in the pedunculopontme nucleus, rostral portson of the lateral parabrachtal nucleus, the dorsal tegmental, medial vestibular (Fig 1J), abducens and preposltus hypoglossal nuclei, the vagal complex, amblguus nucleus and area postrema (Fig 1K) Some scattered cells m the pontlne reticular formation were strongly positive for the a l subunlt In other bramstem regions, expression of the ~1 subumt was weak or very weak On the other hand, the /31 subunlt was strongly expressed m most lower bramstem nuclei (Fig 2) Its expression was strong in the pedunculopontme, pontme, parabrachml, dorsal tegmental, raphe, cochlear, vestibular, facml, abducens, prepos~tus hypoglossal, amblguus and hypoglossal nuclei, reticular formation, nucle~ of the tngemmal nerve, vagal complex and inferior ohve, and
moderate in the other bralnstem nuclei The /31 subunit was weakly expressed in the area postrema, a region in which the c~l subunlt was strongly expressed Cerebellum
Both a l and /31 subunlts were moderately expressed in the Purklnje cell layer (Figs 1 and 2) and weakly expressed in the molecular and granular cell layers, although fllm-autoradlograms displayed high non-speclhc signals m the granular cell layer (Figs I I - K and 2 I - K ) The cerebellar nuclei were strongly posltwe for the /31 subunlt but very weakly positive for the a l subunlt Other regions
a Subumt expression was very strong in the pmeal gland, whereas the/31 subunlt was not detected in this region (Figs 1G,H and 2G,H)
Fig 5 Dark-field photomicrographs of frontal secuons through the h,ppocampus (A,B), paraventrlcular hypothalamlc nucleus (C) and substantla mgra (D) A,C a l subumt B,D fll subumt In the hlppocampus, the /31 subumt is expressed in pyramidal, non-pyramidal, granular and non-granular cells (B) while the a l subuntt is present in non-pyramidal and non-granular cells of the hlppocampus at high levels, In pyramidal cells of the CA2 (2) and CA3 (3) with moderate expression, and with weak expression m pyramidal cells of CA1 (1) and in granular cells in the dentate gyrus (DG) (A) Strong expre,,slon of the a l subumt is noted m the magnocellular subdivision of the paraventrlcular hypothalamlc nucleus (PAm) (B) In the substantia mgra, the/31 subumt is strongly expressed m the pars compacta (SNc) and retlculata (SNr) (D) Moderate to strong expression is also noted m regions adjacent to the substantla ntgra (D) Bar = 100/.Lm
343 DISCUSSION We examined the locahzation of the m R N A of the o<1 and /31 subunlts of SGC in the rat brain by in SltU hybridization using antlsense D N A probes Our findings regarding the/31 subunlt locahzatlon almost corresponded closely with distributions previously determined using immunoh~stochemlstry and in sItu hybridization I'I2'14 For example, we detected very strong expression of the /31 subunlt in the caudate putamen, accumbens nucleus, olfactory tubercle, medial habenula and locus coeruleus, and strong expression in the pyramidal cell layer of the neocortex and hlppocampus, plrlform cortex, subiculum, pontlne nucleus and facial nucleus, and moderate expression m the Purklnje cell layer of the cerebellum In addition to regions reported previously by in SltU hybridization 12, we found strong expression of the /31 subunlt m many nuclei and regions throughout the brain Although Matsuoka et al ~2 found only weak expression of /31 subunlt m R N A in the dlencephalon and lower brainstem other than the pontlne nucleus, locus coeruleus and facial nucleus, we detected moderate to strong expression in many nuclei of the dlencephalon and lower brainstem such as the reticular thalamus, paraventrlcular hypothalamlc nucleus, substantia mgra, dorsal raphe nucleus, red nucleus, cranial motor nuclei, laterodorsal tegmental nucleus, vestibular and cochlear nuclei, and inferior olive The /31 subunlt was expressed throughout the rostrocaudal extent of the brain, with relatively strong expression in the olfactory bulb, many forebraln nuclei, and several bramstem nuclei Our study is the first to examined the locahzatlon of SGC ce subunIt In the brain a l Subunlt expression was also found throughout the brain, and was heterogeneous For example, this subunlt was strongly expressed m the glomerular layer but weakly in other layers of the olfactory bulb, whereas the /31 subunlt was strongly expressed in almost all layers of olfactory bulb In the neocortex, the a l subunlt was expressed strongly in the surface layers, I I / I I I , and weakly m the deeper layers I V - V I , while the /31 subunlt was expressed m both surface and deep layers, I I - V I In the hlppocampus, eel subunlt expression was weak to moderate in pyramidal cells of C A 1 - C A 3 and granular cells of the dentate gyrus, while the/31 subunlt expression was strong in these cells throughout the hlppocampus Isoforms of the /3 subunlt other than /31 have not yet been identified in the rat brain, the recently Identified /32 subumt from the rat kidney 19 is primarily expressed in kidney and hver, but is not expressed in
the brain, while the /33 subunlt cloned from human brain 7 appears to be the human counterpart of the rat /31 subunlt 15 On the other hand, a 2 and a3, lsoforms of the c~1 subunlt, have been cloned from human fetal brain 8 and human adult brain 7, respectively The findlng of wide expression of the /31 subunlt suggests that this subunlt is comprehensive one to form SGC in the brain Harteneck et al 8, using mammalian cells, demonstrated that the expression of ce2 subunlt alone, or the coexpresslon of a 2 subunlt with a l subunlt, did not form active GC, whereas the coexpresslon of a heterodlmer, of a l and /31 subunlts, or of a 2 and /31 subunlts, resulted in the formation of functionally active G C Thus, coexpression of a heterodlmer of a and /3 subunits is necessary for a catalytically active GC, while expression of a homodlmer appears to be insufficient for formation of active G C Nitroprusstde enhanced the activity of heterodlmer GCs expressed in mammalian cells s 13 Coexpression of a l and /31 resulted in an enzyme with higher G C activity than that resulting from coexpresslon of ce2 and /31 subunlts, suggesting that lsoforms of SGC have varying degrees of activity and are probably expressed under differing conditions in the tissues Hence, the various a subunits, together with /31 subunlt, may form lsoforms of active SGC with diverse enzymatic act~wt~es m the rat brain NO, which has recently been shown to be an intercellular biological and neural messenger, activates SGC, and is produced by N O synthase in postsynaptlc and presynaptic structures In response to activation of Nm e t h y l - D - a s p a r t l c acid ( N M D A ) r e c e p t o r s 26 Garthwalte 6 suggested that SGC plays roles m both neuronal and ghal elements adjacent to the neuronal processes producing NO, since SGC lmmunoreactlvlty has been detected in both neuronal and ghal elements 14, high levels of nltroprusslde-sensltlVe G C are present in purified astrocytes obtained from developing cerebellum 4, and mtroprusslde induces accumulation of c G M P in astrocyte cell bodies and processes m slice preparations from young rat cerebellum and in Bergmann ghal cells s 6 On the other hand, we found m R N A of both c~l and /31 SGC subunlts primarily in neuronal cells, suggesting that SGC is stimulated by N O primarily In neuronal elements The localization of SGC m R N A generally overlapped wlth that of N O synthase-containlng fibers, but not necessarily that of cell bodies For example, the caudate putamen cont a m e d numerous neurons that strongly express both subunlts of SGC, and dense networks of N O synthase lmmunoreactlve nerve fibers, while a small number of N O synthase lmmunoreactive neurons were scattered
344 in this region Hence, SGC-labelled cells, which are present at a high density throughout the caudate putamen, do not appear to overlap with NO synthase lmmunoreactlve cells but rather with lmmunoreactlve nerve fibers The cerebellum is another example Purklnje cells were not labeled with N O synthase antisera, but were labelled for SGC, while nerve fibers in the molecular layer and granular cells were positive for NO synthase Thus, SGC in the Purkmje cells and their processes is most hkely activated by N O produced in adjacent nerve fibers in the molecular layer, probably in the parallel fibers In these regions, neurons containing SGC, but not N O synthase, may be targets of NO produced in adjacent neural elements In neurons contamlng both NO synthase and SGC, NO may function as an lntracellular messenger for the activation of SGC Examples Include NO synthasepositive cells in the pedunculopontlne nucleus, which also contains both a l and /31 subunlts of SGC There are some differences between the distributions of SGC and NO synthase One example is the medial habenular nucleus, which strongly express both SGC subunlts but lacks N O synthase positive neurons or terminals In these regions SGC may be stimulated by oxidants other than NO
4cltnowledgrnent This study was supported m part by a Grant-mAid ior Sc,cntff, c Research on Priority Areas (S I and H T) and by grants from Senn Life Science Foundation (S I ) We thank to Mr K Inoue for photographic assistance
REFERENCES 1 A n a n o , M A , Lewlckl J A , Brandwem, H J and Murad, F Immunohlstochemlcal localization of guanylate cyclase within neurons of rat brain, Proc Natl Acad Sct USA 79 (1982) 1316-1320 2 Arnold, W P Mlttal, C K , Katsukl S and Murad F Nitric oxide activates guanylate eyclase and increases guanoslne 3' '~'cyclic monophosphate levels m various tissue preparations, Pro~ Natl Acad Set USA, 74 (1977) 3203-3207 3 Bredt, D S , G l a t t , C E , H w a n g , P M , F o r u h l M Dawson T M and Snyder, S , Nitric oxide synthase protein and m R N A are discretely localized m neuronal populations of the m a m m a h a n
4
5
6 7
8
9
10
II
12
13
14
1~
16
17 18 19
CNS together with N A D P H dlaphorase Neuron 7 (1991) 615624 Bunn S J Garthwalte J and WiIkln, G P , Guanylate cyclase actlvmes m enriched preparations of neurons astrogha and a synaptlc complex isolated from rat cerebellum Neurochem Int 8 (1986) 179-185 de Vente J Bol J G J M and Stembusch H W M ix)cahzatlon of cGMP m the cerebellum of the adult rat an ~mmunohlstochemical study, Bram Rex 504 (1989) 332-337 Garthwmte J~ Glutamate nitric oxide and cell-cell slgnalhng m the nervous system, Trends Neuroscl, 14 (1991) 60-67 Guilt G Scholl, U Bulle F and Guellaen, G , Molecular cloning of the c D N A s coding lor the two subumts of soluble guanylyl cvclase from h u m a n brain, FEBS Lett, 304 (304) 83-88 Harteneck C Wedel, B , Koeshng D , Malkewltz, J , Bohme, E and Schulz, G , Molecular cloning and expression of a new a - s u b u m t ot soluble guanylyl cyclase lnterchangeaNhty of the c~-subunlt of the enzyme, FEBS Lett 292 (1991) 21%222 Kam~sakl, Y , Sahekl S , Nakane, M , Palmlen J A , Kuno, T , Chang, B Y Waldman S A and Murad, F Soluble guanylate cyclase from rat lung exists as a heterodlmer, J Btol C h e m , 261 (1986) 7236-7241 Koeshng, D Harteneck ( Humbert, P Bosst~rhoff, A Frank, R Schultz, G and Bohme E , The primary structure of the larger subumt of soluble guan~lyl cyclase from bovme lung Homology between the two subumts of the enzyme, FEBS Lett 266 (1990) 128-132 Koeshng, D , Hertz, J , Gausepohl, H , Nlroomand, F Hmsch, K - D , Mulsch H Bohme, E Schultz, G and Frank, R The primary structure of the 70 kDa subumt of bovine soluble guanylate cyclase FEB5 Lett 239 (1988) 29-34 Matsuoka I Gmh, G , Poyard, M , Stengel, D Parma, J Guellaen G and H a n o u n e J , Localization of adenylyl and guanvlyl cyclase m rat brain by m sltu hybridization comparison with calmoduhn m R N A distribution J Neurosct 12 (1992) ~350-3~60 Nakane, M Arakl K Sahekl, S Kuno, T Buechler W and Murad, F , Molecular cloning and expression of c D N A s coding tor soluble guanylate cyclase from rat lung, J Btol Chern, 265 (1990) 16841 - 16845 Nakane M Ichtkawa M and Deguchi, T , Light and electron microscopic d e m o n s t r a n o n o1 guanylate cyclase in rat brain, Brain Res, 273 (1983) 9 - 15 Nakane M , Sahekl S Kuno T lshn, K and Murad F Molecular cloning of a c D N A coding lor 70 kllodalton s u b u m t of soluble guanylate cyclase lrom rat lung Btochem Btophvs Res ( o m m u n 157 (1988) 1139-1147 Noguchl, K Monta, Y , Klyama H , Ono, T and Tohyama, M A noxmus stimulus reduces the preprotachlkmms gene expression in the rat, Mol Brain Res, 4 (1988) 31-35 Paxmos, G and Watson, (2 The Rat Bram m 5tereota~:l~ (oord:nares 2nd edn Academic Press, 1986 Schulz, S , Yuen, P S T and Garbers S L , The expanding lamdy of guanylyl cyclases Trends Pharmacol Sct 12 (1991) 116-120 Yuen, P S T , Potter, L R and Garbers, D L A new torm o1 guanylyl cyclase is preferentially expressed in rat kldne~ Btochemtsm,, 29 (1990) 10872-10878
335
BRESM 70669
Localizations of a l and/31 subunits of soluble guanylate cyclase in the rat brain T
Furuyama,
S
Inagakl
and
H
Takag~
First Department of Anatomy, Osaka City Untt,erstty Medical School, Osaka (Japan) (Accepted 4 May 1993)
Key words Soluble guanylate cyclase, Soluble guanylate cyclase a l subunlt, Soluble guanylate cyclase/31 subumt, Rat brain In sltu hybridization
We studied the Iocahzatlons of a l and/31 subumts of soluble guanylate cyclase using m sltu hybridization The/3 subunlt was widely distributed m most neurons throughout the brain, with different levels of expression The ctl subumt was also dl~tnbuted throughout the brain, however, it was located in more hmlted regions Both subunlts were expressed markedly In the glomerular layer of the olfactory bulb, dorsal and ventral strlatum, and several regions in the bralnstem Regions with httle or no a l subumt expression, but with marked expression of the /31 subumt included the olfactory bulb except for the glomerular layer, pyramidal cell layer m CA1 and granular cell layer in the dentate gyrus of the h~ppocampus, and many bramstem nuclei The above regmns expressing both subumts are suggested to contain active soluble guanylate cyclase as a target for mtnc orade, and thus may be revolved m cellular signal transductlon
membrane
INTRODUCTION
a n d a c t i v a t i n g S G C to i n c r e a s e c G M P
fol-
lowing stimulation of excitatory ammo acid receptors Guanylate
cyclases
3',5'-monophosphate
(GCs)
form
cychc
guanoslne
m the bram 6
( c G M P ) , a n m t r a c e l l u l a r signal-
ing m o l e c u l e p r e s e n t in v a r i o u s tissues, i n c l u d i n g t h e b r a i n G C s exist m s o l u b l e a n d m e m b r a n e - b o u n d
S o l u b l e G C ( S G C ) ( E C 4 6 1 2) is r i c h m l u n g a n d b r a i n a n d is a c t w a t e d by n i t r i c o x i d e ( N O ) 26 18 S G C contains heme, a putatwe
MATERIALS AND METHODS
forms
r e c o g n i t i o n s~te f o r N O , a
s t i m u l a t o r for this e n z y m e 6'~8. S G C p u r i f i e d f r o m l u n g h a s a m o l e c u l a r m a s s o f a b o u t 150 k D a a n d exists as a
Animals Five W~star rats weighing 150 g were used Rats were decapitated under sodium pentobarbltal anesthesia (50 mg/kg, i p ) The brams were qmckly removed and frozen in powdered dry ice Serial sectmns m 15/~m thickness were cut on a cryostat, thaw-mounted on sdyated glass slides and kept at -70°C untd they were ready for use for m sltu hybridization
h e t e r o d l m e r o f 82 k D a ( a ) a n d 70 k D a (/3) s u b u m t s 9 Recently, cDNA for each subumt has been cloned f r o m b o v i n e a n d r a t l u n g ~°'t1,~3,a5 E x p r e s s i o n o f o n l y o n e o f t h e s e s u b u m t s in m a m m a h a n cells r e s u l t e d m n o d e t e c t a b l e G C a c t w l t y 13'~5 C o e x p r e s s l o n o f b o t h a a n d / 3 s u b u n l t s a p p e a r s to b e n e c e s s a r y f o r S G C actlvltys ~s A l t h o u g h t h e l o c a h z a t l o n o f t h e /31 s u b u n l t m t h e b r a i n has b e e n
s t u d l e d by m sltu h y b r i d i z a t i o n ,
there are no reports concerning the locahzat~on of the a
subunlt
In the
present
study,
we
examined
the
d i s t r i b u t i o n o f t h e a l a n d /31 s u b u n l t s o f S G C m t h e rat b r a i n W e also a t t e m p t e d to clarify t h e t a r g e t s o f N O , a g a s e o u s m e s s e n g e r c a p a b l e o f c r o s s i n g t h e cell
In sltu hybndtzanon In SltU hybridization was carried out using methods described previously 16 After drying the shde-mounted sections at room temperature, the tissue sections were fixed m 4% paraformaldehyde m 0 1 M phosphate buffer (pH 7 2) for 10 mm, rinsed with phosphatebuffered saline (PBS) three times, and dehydrated in a graded series ot ethanols and chloroform The hybridization was performed overmght at 42°C m a buffer containing 4×SSC, 50% formamlde, 0 12 M phosphate buffer, l×Denhardt's solution, 02% sodmm dodecylsulfate, 250 ~ g / m l yeast RNA, 10% dextran sulfate, 100 mM dlthlothreltol with 106 cpm of labeled probe m 150 p.l buffer per shde Following hybridization, the sections were rinsed m I × S S C three times each for 20 mln at 55°C, this was followed by brief dehydration with 70% and 100% ethanol The tissue sections were then exposed to X-ray film (Kodak, XAR or Amersham, Hyperfilm /3-max) for 1 week to obtain for film autoradlograms, or were coated
Correspondence T Furuyama, First Department of Anatomy, Osaka City Umverslty Medical School, 1-4-54 Asahlmachl, Abeno-ku, Osaka, 545 Japan Fax (81)6-646-6611
336 with lltord K-5 emulMon (diluted 1 1 wtth distilled water at 40°C) and exposed for 4 - 6 weeks at 4°C in light-tight boxes for mlcroautoradiography Following development in D19 (Kodak) the sections were counterstamed with thionm and then observed using dark- and bright-field microscopes (Olympus) Neurons with grain densities at least 3 times higher than the background density were considered positively labelled As a control a competition experiment was done The brain sections were hybridized with labeled ohgonucleottde probes together with 100X
1
excess unlabeled probe No s p e o h c s~gnaN were detected alter the c o m p e t m o n experiment
Ohgonut leottde probe~
Ohgonucleotlde probes were synthesized in an Apphed Blosysterns D N A synthesizer and purified using a N E N S O R B P R E P column (NEN) The a l and /31 subumt probes were complementary to bases 718-766 of the rat SGC (~1 s u b u m t I~ and 575-623 of the rat
al
2
,81 A
I
B
r k UI
r k
v
r ¸
E
r
k
SN
Figs 1 and 2 Fdm autoradlograms showing the dmtnbutlon ol the SGC a l s u b u m t (left side, Fig 1) and /~1 subumt (right side Fig 2) Frontal sections of the rat brain are arranged from rostral to caudal, A - K 3 oculomotor nucleus, 7 facial nucleus, A, accumbens nucleus, AP area postrema, CG cmgulate cortex, CP, caudate putamen, D R , dorsal raphe nucleus LC, locus coeruleus, LD, laterodorsal tegmental nucleus M H medial habenular nucleus OT, olfactory tubercle, P, piriform cortex PB, parabigemInal nucleus, PG pineal gland, R red nucleus RT, reticular thalamlc nucleus, S, subiculum, SC, superior colhculus SN substantla mgra V vestibular nuclei
337 /31 subumt ~5, respectively These ollgonucleotldes were 3'-endlabeled with terminal deoxynucleotldyl transferase (Takara Shuzo) and [a-35S]dATP (NEN) to a specific actwlty of 109 cpm//xg Nomenclature used followed that m the Paxlnos and Watson atlas 17
RESULTS T h e a l s u b u n l t of S G C was p r e s e n t t h r o u g h o u t the b r a i n b u t ~ts expression was h m l t e d to several regions
B1
ctl 1
I:
2
G
H
K
Figs 1 and 2 (continued)
338 (Fig 1), while the /31 subunit of SGC was more widely distributed throughout the brain with differences in the level of the expression (Fig 2) SGC was usually present in neuronal cells (Fig 3) Both of the subunits were expressed abundantly in several forebraln and brainstem regions Furthermore, the /31 subunlt was expressed in many nuclei of the forebraln and brainstem in addition to the regions described above, m which both subunlts were abundant In some regions, such as the area postrema and pineal gland, the expression of the /31 subunlt was very weak, but that of the a l subunlt was strong Table I summarizes the intensities of expression in various regions
strongest expression of the a l subunlt in the rat brain (Fig 1 A - D ) The /31 subunit was also expressed strongly in these regions (Fig 2 A - D ) Numerous neurons expressed strong signals for both subunlt in the caudate putamen and fundus strlatl (Fig 4C,D) The globus palhdus and entopeduncular nucleus exhibited weak to moderate expression for both subunlts on fllm-autoradlograms (Fig 1A-C), while on mlcroautoradlograms there were scattered neurons but each neuron showed moderate to strong expression for both signals (Fig 4C,D) In the cerebral neocortex, the expression of the a l subunit was strong in the surface layer, layers II and III, weak In the deep layer, layers V and VI, and very weak In layer IV (Fig 1) The/31 subunlt was strongly expressed throughout the cortex, with the strongest expression seen in layer V and weak expression in layer IV (Fig 2) Expression of both a l and/31 subunlts was strong in the plrlform cortex and clngulate cortex (Figs l, 2 and 4A) In the clngulate cortex, a 1 expression was strongest in the surface layer at rostral level (Fig 1A,B), while/31 expression was strong in both surface and deep layers (Fig 2A,B) On film-autoradlograms, the density of the /31 subunit was higher than that of the a l subunlt in the hlppocampus (Figs 1D,E and 2D,E) The a l subunlt was strongly expressed in scattered, non-pyramidal and non-granular cells of the hlppocampus, moderately expressed in pyramidal cells of CA2 and CA3, and weakly expressed in pyramidal cells of CA1 and granular cells of the dentate gyrus (Fig 5A) There was also moderate a l subunit expression m the sublculum On the other hand, the /31 subunlt strongly expressed in both pyramidal and non-pyramidal cells of C A 1 - C A 3 , granular and non-granular cells of the dentate gyrus, and in the sublculum (Fig 5B) In the amygdala, expression ot the a l subunlt was
Forebram In the olfactory bulb, the a l subunIt was strongly expressed in the glomerular layer but weakly expressed m the other layers, whereas the /31 subunlt was expressed abundantly In many neurons of most layers of the olfactory bulb including the glomerular, mitral cell and internal granular cell layers In the anterior olfactory nuclei, the expression of the a l subunlt was moderate to strong, and that of the/31 subunlt was strong Neither subunlt was detected in the ependymal and subependymal layers The expression of the a l subunlt was very strong in the olfactory tubercle and accumbens nucleus (Figs 1A, 3 and 4A), strong in the tenla tecta and bed nucleus of the stria termlnahs, and weak to moderate in the septal nuclei and nuclei of the diagonal band Positive neurons were scattered in the substantla lnnomlnata The /31 subunIt was strongly expressed m the regions with moderate to strong expression of the a l subunlt and in the regions such as the islands of Calleja, where the a l subunit was weakly expressed (Figs 2A,B and 4B) The caudate putamen and fundus strlatl showed the
|
)
•
al
i
P
)O Fig 3 Bright-field photomicrographs of the accumbens nucleus Signals for the a l (A) and /31 (B) subumts are present over neuronal cell bodies Bar = 10 p.rn
339 TABLE I
Summary of the mRNA expression of the oil and/31 subuntts of SGC in the rat brain by In sttu hybridization + + + +, very strong, + + +, strong, + +, moderate, +, weak, +, very weak (arbltary umts) al
/31
+ + + + + + +
+ + + +
+ + + +
+ + + +
+ + + + + + + + + + +
+ + + + + + + + + + +
+ + + + + + + + + + +
+ + +
+ + + + + + ___ + +
+ + + + +
+ +
+ + + + + +
+ + + + + +
Hippocampus p y r a m i d a l cells in t h e C A 1 p y r a m i d a l cells in t h e C A 2 p y r a m i d a l cells In t h e C A 3 n o n - p y r a m i d a l cells dentate gyrus sub~culum
+ + + + + +
+ + + + + +
Amygdala lateral nucleus basolateral nucleus intercalated nucleus central nucleus
+ + + + + + +
+ + + + + + +
Thalamus medml habenula reticular thalamlc nucleus ventromedlal thalamlc nucleus zona mcerta parafasc~cular nucleus posterior mtralammar thalamlc nucleus
+ + + + + +
+ + + + + +
+ + + + + + + + + +
Hypothalamus paraventrlcular nucleus magnocellular subdwlslon p a r v o c e l l u l a r subdwls~on supraopt~c nucleus lateral hypothalamlc nucleus
+ + + + + + + +
+ + + +
+ + + + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
Forebram Olfactory bulb glomerular layer m l t r a l cell l a y e r internal granular layer anterior olfactory nucleus tema tecta accumbens nucleus septal nuclei nuclei of the diagonal band olfactory tubercle islands Calleja bed nucleus of the stria termlnahs caudate putamen f u n d u s strmtl globus palhdus entopeduncular nucleus Neocortex l a y e r II l a y e r III layer IV layer V layer VI p~rlform c o r t e x cmgulate cortex
+ + + + +
+ + + + + + + + + + + + +
+ + + + +
+ + + + + + +
+ + +
+ + + + + +
+ + + + + +
Dtencephalon + + + + + + + + + +
Mtdbram substantla mgra pars compacta substantla mgra pars retlculata lnterpeduncular nucleus Darkschewltsch nucleus r e t r o r u b r a l field a n d n u c l e u s raphe nuclei central grey oculomotor nucleus parab~gemmal nucleus
+ + + + + + + + + +
+ +
+ + +
34O TABLE I (continued) c~l
/31
+ + +
I-++ ++
+++
+++
+ ++++ ++++
+++ +++ +++ +++
Mtdbratn
superior colhculus superficial gray layer other layers inferior colhculus
++
Pons and medulla oblongata
pedunculopontme nucleus pontme nucleus laterodorsal tegmental nucleus locus coeruleus lateral parabrachial nucleus dorsal tegmental nucleus raphe nuclei reticular formation cochlear nucle~ vestxbular nuclm racial nucleus abducens nucleus nuclei of the tngemmal complex prepositus hypoglossalnucleus vagal complex amb~guus nucleus hypoglossalnucleus inferior olive area postrema
++ ++ + ++
+++ +++ +++ +++
+ +4
+++ +-++
+ ++
+++ +++
+ ++
+++ +++ +++ +++
++ ++ +
+++
+ +++
+
Cerebellum
Purkmje cells molecular layer granular cell layer cerebellar nuclm
++
++
+ +
+ + +++
Other regions
pineal gland
++++
moderate in the lateral, basolateral, and intercalated amygdaloid nuclei, weak in other amygdalold nuclei, including the basomedlal, medial, anterior and posterior amygdalold nuclei, and very weak in the central amygdalold nucleus /31 Subunlt expression was rather even, being moderate, throughout the amygdala, except in the central amygdaloid nucleus, where the expression was very weak Dtencephalon
Expression of the a l subumt was detected in several limited regions The medial habenula was one of the regions of the brain showing the strongest expression of both a l and 131 subunlts (Figs 1D and 2D) Moderate to strong expression of c~l and /31 subunlts was also noted In the reticular and ventromedial thalamlc nuclei, and in the zona lncerta (Figs 1C,D and 2C,D) The expression of the a l subunit was moderate in the parafasclcular nucleus and posterior lntralamlnar thalamlc nucleus, and weak or very weak in other regions of the thalamus On the other hand, the /31 subunit was distributed more evenly with moderate to strong expression throughout the thalamus, with the expres-
sion being stronger in the reticular and ventromedial thalamlc nuclei, and relatively weak in the ventral posterolateral and ventral posteromedlal thalamic nuclei The pretectal nuclei showed weak to moderate expression of the a l subunlt and moderate to strong expression of the/31 subunlt In the hypothalamus, a l subunlt was strongly expressed in the magnocellular subdivision of the paraventrlcular hypothalamlc (Fig 5C) and supraoptic nuclei, whereas it was weakly expressed in other hypothaiamlc nuclei In contrast, the/31 subunlt expression was more even, and moderate in most hypothalamlc nuclei, with relatively stronger expression m the magnocellular and parvoceUular subdivisions of the paraventricular nucleus and in the lateral hypothalamlc nucleus Mtdbram
Expression of the a l subunit was strong in the oculomotor nucleus, parablgemlnal nucleus (Fig 1G), and raphe nuclei including the dorsal raphe nucleus (Fig IG), moderate in the interpeduncular and Darkschewitsch nuclei, central gray and retrorubral field and nucleus, and weak in other mldbraln regions in-
341 c l u d l n g t h e s u p e r i o r a n d i n f e r i o r c o l h c u h ( F i g 1) S c a t t e r e d n e u r o n s m t h e d e e p m e s e n c e p h a h c nucleus w e r e m o d e r a t e l y positive for t h e a l s u b u n t t B e t a 1 s u b u n i t e x p r e s s i o n was m o r e even, b e m g m o d e r a t e to s t r o n g e x p r e s s i o n t h r o u g h o u t t h e m l d b r a l n , with relattvely s t r o n g e x p r e s s i o n m t h e s u b s t a n t l a m g r a p a r s c o m p a c t a a n d r e t l c u l a t a (Figs 2E a n d 5 D ) a n d the b o t t o m layer o f t h e superficial gray l a y e r o f t h e s u p e -
al
r l o r colhculus ( F i g 2 E - G ) , in a d d i t i o n to the nuclei exhibiting m o d e r a t e to s t r o n g a l e x p r e s s i o n
Pons and medulla oblongata T h e e x p r e s s i o n o f the /31 r a t h e r even, b e i n g m o d e r a t e to pons and medulla oblongata, s u b u n t t was h e t e r o g e n e o u s a n d
s u b u m t was g e n e r a l l y strong, t h r o u g h o u t the whtle t h a t of the a l h m i t e d to several nu-
.6'1
Fig 4 Dark-field photomicrographs showlrig the a l (A,C,E) and /31 (B,D,F) subumts m regions In which both subunlts are strongly expressed Frontal sections through the olfactory tubercle (A,B), caudate putamen (C,D) and locus coeruleus (E,F) Strong or very strong expression of the a l and/31 subunlts Is seen m the olfactory tubercle (OT) and preform cortex (P), caudate putamen (CP) and locus coeruleus (LC), and moderate expression is observed m scattered cells in the globus palhdus (GP), reticular thalamlc nucleus (RT) and raphe pontls (RP) Bars m A - E = 100 g.m, m F = 100 p.m
342 clear groups The laterodorsal tegmental nucleus and locus coeruleus very strongly expressed both a l and/31 subunlts (Figs 1H,I, 2H,I and 4E,F) In addition, the expression of the a l subunlt was moderate to strong in the pedunculopontme nucleus, rostral portson of the lateral parabrachtal nucleus, the dorsal tegmental, medial vestibular (Fig 1J), abducens and preposltus hypoglossal nuclei, the vagal complex, amblguus nucleus and area postrema (Fig 1K) Some scattered cells m the pontlne reticular formation were strongly positive for the a l subunlt In other bramstem regions, expression of the ~1 subumt was weak or very weak On the other hand, the /31 subunlt was strongly expressed m most lower bramstem nuclei (Fig 2) Its expression was strong in the pedunculopontme, pontme, parabrachml, dorsal tegmental, raphe, cochlear, vestibular, facml, abducens, prepos~tus hypoglossal, amblguus and hypoglossal nuclei, reticular formation, nucle~ of the tngemmal nerve, vagal complex and inferior ohve, and
moderate in the other bralnstem nuclei The /31 subunit was weakly expressed in the area postrema, a region in which the c~l subunlt was strongly expressed Cerebellum
Both a l and /31 subunlts were moderately expressed in the Purklnje cell layer (Figs 1 and 2) and weakly expressed in the molecular and granular cell layers, although fllm-autoradlograms displayed high non-speclhc signals m the granular cell layer (Figs I I - K and 2 I - K ) The cerebellar nuclei were strongly posltwe for the /31 subunlt but very weakly positive for the a l subunlt Other regions
a Subumt expression was very strong in the pmeal gland, whereas the/31 subunlt was not detected in this region (Figs 1G,H and 2G,H)
Fig 5 Dark-field photomicrographs of frontal secuons through the h,ppocampus (A,B), paraventrlcular hypothalamlc nucleus (C) and substantla mgra (D) A,C a l subumt B,D fll subumt In the hlppocampus, the /31 subumt is expressed in pyramidal, non-pyramidal, granular and non-granular cells (B) while the a l subuntt is present in non-pyramidal and non-granular cells of the hlppocampus at high levels, In pyramidal cells of the CA2 (2) and CA3 (3) with moderate expression, and with weak expression m pyramidal cells of CA1 (1) and in granular cells in the dentate gyrus (DG) (A) Strong expre,,slon of the a l subumt is noted m the magnocellular subdivision of the paraventrlcular hypothalamlc nucleus (PAm) (B) In the substantia mgra, the/31 subumt is strongly expressed m the pars compacta (SNc) and retlculata (SNr) (D) Moderate to strong expression is also noted m regions adjacent to the substantla ntgra (D) Bar = 100/.Lm
343 DISCUSSION We examined the locahzation of the m R N A of the o<1 and /31 subunlts of SGC in the rat brain by in SltU hybridization using antlsense D N A probes Our findings regarding the/31 subunlt locahzatlon almost corresponded closely with distributions previously determined using immunoh~stochemlstry and in sItu hybridization I'I2'14 For example, we detected very strong expression of the /31 subunlt in the caudate putamen, accumbens nucleus, olfactory tubercle, medial habenula and locus coeruleus, and strong expression in the pyramidal cell layer of the neocortex and hlppocampus, plrlform cortex, subiculum, pontlne nucleus and facial nucleus, and moderate expression m the Purklnje cell layer of the cerebellum In addition to regions reported previously by in SltU hybridization 12, we found strong expression of the /31 subunlt m many nuclei and regions throughout the brain Although Matsuoka et al ~2 found only weak expression of /31 subunlt m R N A in the dlencephalon and lower brainstem other than the pontlne nucleus, locus coeruleus and facial nucleus, we detected moderate to strong expression in many nuclei of the dlencephalon and lower brainstem such as the reticular thalamus, paraventrlcular hypothalamlc nucleus, substantia mgra, dorsal raphe nucleus, red nucleus, cranial motor nuclei, laterodorsal tegmental nucleus, vestibular and cochlear nuclei, and inferior olive The /31 subunlt was expressed throughout the rostrocaudal extent of the brain, with relatively strong expression in the olfactory bulb, many forebraln nuclei, and several bramstem nuclei Our study is the first to examined the locahzatlon of SGC ce subunIt In the brain a l Subunlt expression was also found throughout the brain, and was heterogeneous For example, this subunlt was strongly expressed m the glomerular layer but weakly in other layers of the olfactory bulb, whereas the /31 subunlt was strongly expressed in almost all layers of olfactory bulb In the neocortex, the a l subunlt was expressed strongly in the surface layers, I I / I I I , and weakly m the deeper layers I V - V I , while the /31 subunlt was expressed m both surface and deep layers, I I - V I In the hlppocampus, eel subunlt expression was weak to moderate in pyramidal cells of C A 1 - C A 3 and granular cells of the dentate gyrus, while the/31 subunlt expression was strong in these cells throughout the hlppocampus Isoforms of the /3 subunlt other than /31 have not yet been identified in the rat brain, the recently Identified /32 subumt from the rat kidney 19 is primarily expressed in kidney and hver, but is not expressed in
the brain, while the /33 subunlt cloned from human brain 7 appears to be the human counterpart of the rat /31 subunlt 15 On the other hand, a 2 and a3, lsoforms of the c~1 subunlt, have been cloned from human fetal brain 8 and human adult brain 7, respectively The findlng of wide expression of the /31 subunlt suggests that this subunlt is comprehensive one to form SGC in the brain Harteneck et al 8, using mammalian cells, demonstrated that the expression of ce2 subunlt alone, or the coexpresslon of a 2 subunlt with a l subunlt, did not form active GC, whereas the coexpresslon of a heterodlmer, of a l and /31 subunlts, or of a 2 and /31 subunlts, resulted in the formation of functionally active G C Thus, coexpression of a heterodlmer of a and /3 subunits is necessary for a catalytically active GC, while expression of a homodlmer appears to be insufficient for formation of active G C Nitroprusstde enhanced the activity of heterodlmer GCs expressed in mammalian cells s 13 Coexpression of a l and /31 resulted in an enzyme with higher G C activity than that resulting from coexpresslon of ce2 and /31 subunlts, suggesting that lsoforms of SGC have varying degrees of activity and are probably expressed under differing conditions in the tissues Hence, the various a subunits, together with /31 subunlt, may form lsoforms of active SGC with diverse enzymatic act~wt~es m the rat brain NO, which has recently been shown to be an intercellular biological and neural messenger, activates SGC, and is produced by N O synthase in postsynaptlc and presynaptic structures In response to activation of Nm e t h y l - D - a s p a r t l c acid ( N M D A ) r e c e p t o r s 26 Garthwalte 6 suggested that SGC plays roles m both neuronal and ghal elements adjacent to the neuronal processes producing NO, since SGC lmmunoreactlvlty has been detected in both neuronal and ghal elements 14, high levels of nltroprusslde-sensltlVe G C are present in purified astrocytes obtained from developing cerebellum 4, and mtroprusslde induces accumulation of c G M P in astrocyte cell bodies and processes m slice preparations from young rat cerebellum and in Bergmann ghal cells s 6 On the other hand, we found m R N A of both c~l and /31 SGC subunlts primarily in neuronal cells, suggesting that SGC is stimulated by N O primarily In neuronal elements The localization of SGC m R N A generally overlapped wlth that of N O synthase-containlng fibers, but not necessarily that of cell bodies For example, the caudate putamen cont a m e d numerous neurons that strongly express both subunlts of SGC, and dense networks of N O synthase lmmunoreactlve nerve fibers, while a small number of N O synthase lmmunoreactive neurons were scattered
344 in this region Hence, SGC-labelled cells, which are present at a high density throughout the caudate putamen, do not appear to overlap with NO synthase lmmunoreactlve cells but rather with lmmunoreactlve nerve fibers The cerebellum is another example Purklnje cells were not labeled with N O synthase antisera, but were labelled for SGC, while nerve fibers in the molecular layer and granular cells were positive for NO synthase Thus, SGC in the Purkmje cells and their processes is most hkely activated by N O produced in adjacent nerve fibers in the molecular layer, probably in the parallel fibers In these regions, neurons containing SGC, but not N O synthase, may be targets of NO produced in adjacent neural elements In neurons contamlng both NO synthase and SGC, NO may function as an lntracellular messenger for the activation of SGC Examples Include NO synthasepositive cells in the pedunculopontlne nucleus, which also contains both a l and /31 subunlts of SGC There are some differences between the distributions of SGC and NO synthase One example is the medial habenular nucleus, which strongly express both SGC subunlts but lacks N O synthase positive neurons or terminals In these regions SGC may be stimulated by oxidants other than NO
4cltnowledgrnent This study was supported m part by a Grant-mAid ior Sc,cntff, c Research on Priority Areas (S I and H T) and by grants from Senn Life Science Foundation (S I ) We thank to Mr K Inoue for photographic assistance
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