MOLE(LII.

AR

AND

CELLILAR

iiEI~KOS(IENCES

1,

161-167

(1990,

Autoradiographic Localization of Neuromedin Binding Sites in Rat Brain

Received

for phlication

.lune

20. 1990

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__-___.__

The binding properties and autoradiographic distribution of radiolabeled neuromedin B (NMB) to rat brain sections were investigated. lz51-NMB bound with high affinity (K, = 1 nM) to a single class of sites (B,,, = 80 fmol/mg protein) in coronal rat brain sections. Binding was specific, time dependent, and reversible. Structure activity studies indicated that NMB, litorin, ranatensin, bombesin, gastrin releasing peptide (GRP), and GRP1a27 12I-NMB binding with high but not GRP- inhibited affinity. Autoradiographic studies indicated that high 251-NMB grain densities were present in the olfactory bulb, nucleus accumbens, and central medial thalamic nucleus. Moderate grain densities were present in the septum, stria terminalis, hypothalamus, hippocampus, amygdala, locus coeruleus, dorsal parabrachial nucleus, and superior olive. Grains were absent in the corpus callosum, lateral thalamus, and cerebellum. Because NMB binds with high affinity to discrete brain regions, it may function as a regulatory peptide in the rat central nervous system. c 1990 Academic Press, Inc.
._--__-----_~

binding with high affinity and GRP as well as BN stimulates phosphatidylinositol turnover (6, 7). Also, BN and GRP are potent grooming and satiety agents when injected into the rat CNS (8, 9). These data indicate that GRP may function as a neuromodulatory agent in the rat CNS. NMB, which is structurally similar to GRP, may function as a unique CNS peptide. The gene for rat and human NMB is distinct from that of GRP (10). Human NMB contains a signal peptide followed by NMB and a NMB gene-associated peptide (11). NMB is present in synaptosomes and is released in a Ca -dependent manner (12). NMB is localized to unique regions of the rat brain such as the olfactory bulb and the hippocampus (13-15). Receptors for NMB and the biological activities of NMB in the CNS have not been characterized. Here binding sites for 1-NMB were characterized and localized to discrete rat brain regions using in vitro autoradiographic techniques. METHODS (Tyr)NMB was synthesized using standard solid-phase procedures described previously (16). Purity was assessed by HPL( and amino acid analysis and was greater than 97%. iTyr)NMB was iodinated using the iodogen pro. cedure. Iodogen (0 4 $1~) was added to 8 pg of (Tyr)NMB dnd :! mCi ?;aiLI in (!.5 d! NaHPO, (pH 7.4) at room temperature. After 6 min 300 ~1 of II,0 was added and the free iodide removed. Specifically, the reaction mixture was loaded onto a C,, Sep-Pak using 0.25 M triethylammonium phosphate (TEAP), pH 3.5. The Sep-Pak was rinsed with 5 ml of H,O and the radiolabeled peptide eluted with TEAP/actonitrile (l/l). The radiolabeled peptide was diluted in HZ0 and loaded onto the HPLC containing a C,, Bondapak resin. (51-Tyr0)NMB was eluted using a 0.1% TFA/GO% acetonitrile gradient and the specific activity was 2200 Ci/mmol. The I-NMB analogue was then used in receptor binding studies. Sections of fresh frozen rat brain (20 pm

INTRODUCTION The hombesin (HN) family of peptides is biologirall) active in the mammalian CNS. These peptides include :he 27 amino acid gastrin releasing peptide (GRP1 and I he Ii) amino acid neuromedin B (NMB) which have Y of the same 10 C-terminal amino acids (Table 1). GRP immunoreactivit,y is localized to certain areas of the rat brain, including the stria terminalis, paraventricular nucleus of the hypothalamus, central gray, and nucleus tractus solitarious (I, 2). The genes for both human and rat GRP have been cloned (3, 4) and human type 1 preproGRP contains a signal sequence followed by GRP and then a GRP gene-associated peptide (GGAP). GRP can be further metabolized to the 10 amino acid GRPIRzi (neuromedin C) which is also biologically active. GRP is localized to synaptosomes and released in a Ca+-dependent manner (5). GRP inhibits specific (I-Tyr4)BN

162

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AI,.

TABLE

1
of

Structure of the BN Family

Peptide GRP BN Lit orin Hanatensin NMB Note. Sequence homologies relative to NMB are underlined.

Neuropeptides

Ala-Pro~Val-Ser~Val-GIyGly-Gly-Gly-Thr~Val-Leu~Ala-Lys -Met~Tyr-Pro-Arg-Gly-Asn-HisTrp-Ala-Val-Gly-His-Leu-Met-NH, Pyr~Gln-Arg-Leu-Gly~Asn-GlnTrp-Ala-Val~Gly-His-Leu-Met-NH, Pyr-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH, Pyr-Val-Pro-Gln-Trp-Ala-VallGly-His-Phe-Met-NH, Gly-Asn-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH,

thick) were sectioned on a cryostat and placed onto glass slides or coverslips. Sections were air-dried and incubated with assay buffer composed of 130 mM NaCl, 5 mM MgCl,, 5 mM KCl, 1 mM MnC12, 1 mM EGTA, 100 pg/ ml bacitracin, and 0.1% bovine serum albumin in 10 mM Hepes . NaOH (pH 7.4) plus radiolabeled peptide at 22C in the presence or absence of competitor (1 ph4 NMB). After incubation, free radiolabeled peptide was removed by two consecutive washes in buffer at 4C followed by a brief rinse in H20. The sections on coverslips, which contained bound peptide, were crushed and assayed for radioactivity, or apposed to H-hyperfilm which contained an 1251-microscale standard (Amersham Corp.). After 2 weeks the film was developed and the grain density analyzed using a RAS 3000 densitometer (Amersham Corp.). RESULTS I-NMB bound with high affinity to coronal rat brain sections which contained the nucleus accumbens. Figure 1 shows that total binding increased rapidly during the

first 30 min, reaching equilibrium (approximately 10,000 cpm) after 60 min. Nonspecific binding was approximately 2000 cpm and the difference between the two represents specific binding which increased rapidly during the first 30 min, reaching equilibrium after 60 min. The ratio of specific/nonspecific binding was 5/l. For all subsequent experiments a 60-min incubation period was used. The dissociation of bound NMB was investigated (Fig. 2). I-NMB slowly dissociated from the sections after addition of 1 pM unlabeled NMB. The half time of dissociation was 60 min and after 180 min 75% of the bound I-NMB dissociated. The rate of I-NMB dissociation was approximately lo-fold slower at 4C than at 24C (data not shown). Specific 125 I- NMB binding was a function of NMB concentration (Fig. 3). At low peptide concentrations such binding increased greatly, as 1 nM, specific I-NMB whereas at high ligand concentrations, e.g., 5 nM specific 251-NMB, binding increased slightly. The binding appeared saturable. A Scatchard plot of the specific binding bound with high affinity data indicated that I-NMB

L
Time, min

I
60

I
120

I
160

Time,

min

Association with coronal rat brain unlabeled NMB and time. The mean value difference between the

FIG. 1.

kinetics. %NMB (0.48 nM) was incubated slices in the presence (0) or absence (0) of 1 pM the amount bound determined as a function of k SE of three determinations is indicated. The two represents specific binding (A).

FIG. 2. Dissociation kinetics. *%NMB (0.36 nM) was incubated with rat brain slices for 60 min and then 1 pM unlabeled NMB was added. The amount of specifically bound *%NMB was determined as a function of time. The mean value of three determinations is indicated.

NEIIROMEDIN

B BINDIN(:

SIIES

FIG. 3. centration. bound was tion. The difference chard plot

Amount of J-NMH hound as a function of peptide conLet?: lhe amount of total (ai and nonspecific (A) I-NMB determined as a function of radiolabeled peptide concentramean value t SE of three determinations is indicated. The between the two represents specific hinding (0). Right: Scatof the Qpecitic binding data.

FIG. 4. Specificity of Iyi I-NMB bmding. The Iwrcentage 2I-NMH hound was determined as a function otunlaheled NMH (O), litorin tt3). GRP (A), HN (3), and GRP (m) using 0.48 nhl I-UMB. The mean value ? SE of tour determinations is indiwtr+

(& = 1 nM) to a single class of sites (R,,;,, = 80 fmol/mg protein). The specificity of binding was investigated. Figure 4 shows that little specific binding was inhibited by 0.1 nM GRP whereas most specific binding was inhibited by 1000 nM GRP. The IC,,, for GRP was 60 nM. In contrast NMB was more potent with an IC,,, value of 4 nM whereas GRP was less potent with an IC,,, value of >lO,OOO nM. Also, GRP 7, ranatensin, litorin, and BN had I&,, values of 6, 20. 20, and 50 nM. The distribution of %NMB binding sites was investigated using in vitro autoradiographic techniques and film grain concentrations were analyzed on the densitometer. High grain densities (5.9 fmol I-NMB/mg protein; Table 2) were present in t,he anterior olfactory nucleus but not control sections treated with 1 pM unlabeled NMB (Fig. 5A, 5G). Also, moderate grain densities were present in the external plexiform layer and internal granular layer of the olfactory bulb (Fig. 5A). High grain densities were present in the olfactory tubercle whereas moderate grain densities were present in the claustrum (Fig. 5B). Figure 5C shows that high I-NMB grains were present in the nucleus accumbens (2.4 fmol/mg) whereas moderate grain densities were present in the cingulate cortex and septal hippocampal nucleus (1.6 fmol/mg). Moderate grain densities were present in the neocortex, especially layers V and VI, whereas the densities in layers I-IV were somewhat lower (Fig. 5D). Also, moderate grain densities were present in the anterior commissure (Fig. 5D) and low densities in the caudate putamen (0.5 fmol/mg). Moderate grain densities were also present in the bed nucleus of the stria terminalis (1.6 fmol/mg), septal hypothalamic nucleus, suprachiasmatic nucleus, and medial preoptic area whereas low grain densities were present in the lateral preoptic area and grains were absent in the corpus callosum (Fig. 5E). Low grain densities were present in the globus pallidus and subfornical organ (Fig. 5E). High I-

NMB (3.5 were clei, and

grains were present in the central medial thalamic fmol/mg) nucleus, whereas moderate grain densities present in the rhomboid and reunllens thalamic nuparaventricular hypothalamic nucleus (0.7 fmol/mg), central amygdaloid nucleus (1 .O fmol!mg). Moderate

TABLE I-NMH (irain Density

2 in Rat Brain Regions 1)ensit.v 11rnol/mg protein 5.88 :~.?x :!.-I:! 1 .fi:! I .m 1.4:1 I .oo 0.98 O.R9 0.81 0.80 0.70 0.68 0.67 o.r,:! 0.48 O.l(i 0. 11 ~*- 0.29 i 0.89 + 0. I!) -t 0.25 2 0. 13 I o.1:i t 0. IO - 0. I2 + 0.06 I 0.07 t 0.1 I
l 0.05

Brain

region.

coordinates

Anterior olfactory nucleus. 14.2 mm Central medial thalamic nucleus. 7.2 mm Nucleus accumbens. 10.2 mm Septal hippocampal nucleus. IO.2 mm Stria terminalis, 8.7 mm Hippocampus. 7.2 mm Superior olive, 0.3 mm Central amygdaloid nucleus, 7.2 mm Ventral medial hypothalamic nucleus, 5.7 mm Medial preoptic nucleus, 8.7 mm Medial amygdaloid nucleus, 5.7 mm Dorsal parahrachial nucleus. 0.3 mm Parventricular hypothalamic nucleus. 7.2 mm Locus roeruleus, -0.3 mm Entorhinal cortex. 0.7 mm Caudate putamen. 9.2 mm Central pray, 2.2 mm Subiculum, 4.2 mm Nucleus t ractus solitarious. 3.X mm Parietal cortex. 8.7 mm

i f i2 + i

0.06 0.09 0.17 0.06 0.10 0.09

0. 11 OF 0.07

0.38 7 0.06

Note. The autoradiographic films were compared to sl-microscale standards on an Amersham RAS 3000 densitometer and the grain density was calculated. The nonspecific binding was subt ratted from the total binding to yield the specific binding data. The mean value + SE of six determinations is indicated when the concentration of I-NMH wa> 90 PM.

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g sites was determined using coronal FIG. 5. Autoradiography of ?-NMB in the rat forebrain. The distribution of total *I-NMB binc m, (E) 8.7 mm, and (F) 7.2 mm. Also, slices derived from Paxinos and Watson (17) coordinates (A) 14.2 mm, (B) 11.2 mm, (C) 10.2 mm, (D) 9.2 sed: anterior olfactory nucleus (AO), nonspecific binding was determined using slices derived from coordinates (G) 14.2 mm. Abbreviations claustrum (Cl), olfactory tubercle (Tu), septohippocampal nucleus (SHi), nucleus accumbens (Acb), cingulate cortex (ACg), anterior commisure (ac), neocortex (Neo), parietal cortex (FrPa), septal hypothalamic area (SHY), bed nucleus of the stria terminalis (BST), medial preoptic (MPO) hippocampus (Hi), dentate gyrus (DG), central medial thalamic nucleus (CM), rhomboid thalamic nucleus (Rh), paraventricular nucleus of the hypothalamus (Pa) and central amygdaloid nucleus (Ce).

grain densities were present in the polymorphic layer of the dentate gyrus as well as the oriens layer, stratum radiatum, and the molecular layer of the hippocampus (1.4 fmol/mg) whereas grains were absent from the lateral thalamus. High grain densities were present in the central medial thalamic nucleus (Fig. 6A) and moderate densities in the

ventral medial hypothalamic nucleus (0.9 fmol 1251-NMB bound/mg protein) and medial amygdaloid nucleus. Figure 6B shows that low grain densities were present in the subiculum (0.4 fmol/mg) and parietal cortex (0.4 fmol/ mg). Moderate and low lz51-NMB grains were present in the pontine reticular nucleus (Fig. 6C) and central gray (0.5 fmol/mg), respectively. Also, low grain densities were

NEIJROMEDIN

B BINDIN(:

SITES

165

FIG. 6. Regional distrihution of I-NMB binding sites. I-NMB autoradiography was determined using coronal slices derived from Paxinos and Watson coordinates (A) 5.7 mm, (B) 4.2 mm, (C) 2.2 mm, (D) 0.7 mm, (E) -0.3 mm, and (F) -3.8 mm. Abbreviations used: hippocampus (Hi), central medial thalamic nucleus (CM), medial amygdaloid nucleus (Me), ventromedial hypothalamus (VMH), suhirulum (S), parietal cortex (FrPa), central gray (CG), pontine nucleus (Pn), dorsal raphe (DR), entorhinal cortex (Ent), inferior colliculus (IC). locus (w>rIIIeus (LX), dorsal parahrachial nucleus (DPB), superior olive (SO), nucleus of the solitary tract (Sol). and cerebellum (Cer).

present in the substantia nigra reticular, ventral tegmental area, medial geniculate nucleus, and superior colliculus (Fig. 6C). Moderate grain densities were present in the dorsal raphe (Fig. 6D) and low grain densities in the entorhinal cortex (0.5 fmol/mg). Moderate grain densities were present in the superior olive (1.0 fmol/mg), locus coeruleus (0.7 fmol/mg), and dorsal parabrachial nucleus (0.7 fmol/mg) whereas low grain densities were present in the inferior colliculus and grains were absent from the pontine reticular nucleus (Fig. 6E). Low grain densities were present in the nucleus of the solitary tract (0.4 fmol/ mg) whereas grains were absent from the parvocellular reticular nucleus and the cerebellum (Fig. 6F).
DISCUSSION

For several classes of peptides, such as CCK and opioids, there are multiple classes of receptors (18, 19). For the BN family of peptides it was recently proposed that there may be two classes of receptors (20). The GRP

receptor, which is present on pancreatic acinar, pituitary, Swiss 3T3, and SCLC cells, binds BN and GRP with high affinity and NMB with low affinity (21 24). The NMB receptor which is present on esophageal muscularis mucosa and gastric smooth muscle cells binds NMB with high affinity and GRP with low affinity (25). Previously, the binding properties of (I-Tyr4)BN to rat brain were determined (26). Here we investigated the binding properties and autoradiographic localization of I-NMB to the rat brain. Using coronal sections which contained the nucleus accumbens, I-NMB bound with high affinity. Binding was time dependent and 1-NMB bound with high affinity (& = 1 nM) to a single class of sites (B,,,, = 80 fmol/mg protein). Binding of 12I-NMB was strongly inhibited by NMB, BN, GRP, ranatensin, and litorin i:Cno values of 4, 50, 60, 20, and 20 nA4, respectively) but, not GRP Ii. Because NMB, BN, and GRP but not GRP-le bind with high affinity to the specific I-NMB binding sites, the C-terminal may be essential for high affinitv binding ac-

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tivity. Previously, we found that the C-terminal octapeptide of BN or GRP was essential for high affinity (lz51Tyr*)BN binding activity (26). The I-NMB binding sites were discretely distributed in the rat brain. High grain densities of lz51-NMB were present in the anterior olfactory nucleus and central medial thalamic nucleus and moderate densities in the hippocampus and locus coeruleus. Because each of these brain regions has a high density of NMB, the iI-NMB binding sites may be activated by endogenous NMB. Moderate grain densities were present in the stria terminalis, paraventricular hypothalamic nucleus, and central as well as medial amygdaloid nuclei. Because these areas have a high density of GRP but not NMB, these lz51-NMB binding sites may be activated by endogenous GRP. The distribution of I-NMB binding sites is surprisingly similar to that of (251-Tyr4)BN. In particular, the density of (I-Tyr4)BN grains was high in the nucleus accumbens where BN alters dopamine turnover (27), and high in the hypothalamus where direct injection of BN causes hypothermia (28), satiety (9), and altered gastric acid secretion (29). Also, the density of (1-Tyr*)BN grains is moderate in cortex where BN stimulates phosphatidylinositol turnover (7), moderate in the hippocampus where BN is an excitatory agent (30), and low in the central grey where BN is an analgesic (31). The biological activities of NMB in the CNS have not been investigated. Previously, it was found that guinea pig pancreatic acini, pituitary, Swiss 3T3, and human glioblastoma cell lines bind (Tyr*)BN (& = 4 nM) but not NMB (& = 100 nM) with high affinity. Preliminary data (Lee et al., unpublished) indicate that certain hypothalamic nuclei bind NMB with approximately lo-fold lower affinity than does the nucleus accumbens. It is possible that these regions contain a unique receptor subtype which prefers BN and GRP relative to NMB. Alternatively, there may be more enzymes, such as aminopeptidases, which degrade NMB more readily than BN or GRP. In Swiss 3T3 and human glioblastoma cells, I-GRP is cross-linked to a glycoprotein of 75 kDa (32). Recently, the Swiss 3T3 GRP receptor was cloned and found to comprise 384 amino acids and 7 hydrophobic domains (33). Thus the GRP receptor has a structure reminiscent of other G-proteincoupled receptors such as the substance K receptor (34). In the superior olive, however, moderate densities of I-NMB but not (iz51-Tyr4)BN grains are present. Currently, we are investigating if NMB binds with higher affinity than does BN or GRP in this brain region. It is possible that in this brain region, similar to the esophageal muscularis mucosa, there is a receptor subtype which prefers NMB relative to BN or GRP. The structure of the NMB receptor remains to be determined. In summary, because 1251-NMB binds with high affinity in the rat CNS to distinct brain regions, NMB may function as a regulatory peptide in the mammalian brain.

ACKNOWLEDGMENTS
The authors thank Rena Gets for helpful discussions. is supported in part by NSF Grant BNS-8815133. This research

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