autoradiografia localizacao nmb

7/28/2019 Autoradiografia Localizacao Nmb

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MOLE(LII.AR AND CELLILAR iiEI~KOS(IENCES 1, 161-167 (1990,

Autoradiographic Localization of Neuromedin BBinding Sites in Rat Brain

Received for phlication . lune 20. 1990

--- __-___.__The binding properties and autoradiographic distri-

bution of radiolabeled neurom edin B (NMB ) to rat brainsections were investigated. lz51-NMB bound with highaff ini ty (K, = 1 nM) to a single class of si tes (B,,, = 80fmo l/mg protein) in coronal rat brain section s. Bindingwas speci f ic, t ime dependent, and reversible. Structureactivi ty studies indicated that NM B, l i tor in, ranatensin,bombesin, gastr in releasing peptide (GRP), and GRP1a27but not GRP - inhibited 12I-NMB binding with highaffinity . Autoradiograph ic studie s indicated tha t high251-NMB grain densi t ies were present in the ol factorybulb, nucleus accumbe ns, and central medial thalamicnucleus. Moderate grain densi t ies were present in theseptum , str ia terminal is, hypothalamus, hippocampus,amygdala, locus coeruleus, dorsal parabrachial nucleus,and superior olive. Grains were absen t in the corpus cal-losum, lateral thalamus, and cerebel lum. Because NMBbinds with high aff ini ty to discrete brain regions, i t mayfunction as a regulatory peptide in the rat central nervoussys tem. c 1990 Academic Press, Inc..- _--__-----_~

IN T R OD U C T IONThe hom besin (HN) fam ily of peptides is biologirall)

active in the mamm alian CNS. These peptides include:he 27 amino acid gastr in releasing peptide (GRP1 andI h e Ii) amino acid neuromedin B (NMB) which have Y ofthe same 10 C-terminal amino acids (Table 1). GRP im-muno reactivi t ,y is local ized to certain areas of the ratbrain, including the stria termina lis, paraven tricular nu-cleus of the hypothalamus, central gray, and nucleustractus sol i tar ious ( I, 2) . The genes for both human andrat GRP have been cloned (3, 4) and human type 1preproGRP contains a signal sequence fol lowed by GRPand then a GRP gene-associated peptide (GGAP). GRPcan be fur ther metabol ized to the 10 amino acid GRPIR zi(neuromedin C) which is also biological ly active. GRP islocal ized to synaptosom es and released in a Ca+-depen-dent manner (5) . GRP inhibits speci f ic ( I-Tyr4)BN

binding with high aff ini ty and GRP as wel l as BN stim-ulates phosphatidyl inosi tol turnover (6, 7) . Also, BN andGRP are potent grooming and satiety ag ents when in-jected into the rat CNS (8, 9) . These data indicate thatGRP may function as a neuromodulatory agent in therat CNS.

NMB , which is structural ly simi lar to GRP, ma y func-t ion as a unique CNS peptide. The gene for rat and humanNMB is dist inct from that of GRP (10). Human NMBcontains a signal peptide fol lowed by NMB and a NMBgene-associated peptide (11). NMB is present in synap-tosome s and is released in a Ca -dependent manner (12).NMB is local ized to unique regions of the rat brain suchas the ol factory bulb and the hippocampus (13-15). Re-ceptors for NMB and the biological activ i t ies of NMB inthe CNS have not been character ized. Here binding si tesfor 1-NMB were character ized and local ized to discreterat brain regions using in vitro autoradiographic tech-niques.

MET H OD S(Tyr)NMB was synthesized using standard sol id-phase

procedures descr ibed previously (16). Pur i ty was assessedby HPL( and amino acid analysis and was greater than97%. iTyr)NMB was iodinated using the iodogen p ro.cedure. Iodogen (0 4 $1~) was added to 8 pg of (Tyr)NMBdnd :! mCi ?;aiL I in ( !.5 d! NaHPO , (pH 7.4) at roomtemperature. After 6 min 300 ~1 of I I ,0 was added andthe free iodide removed . Speci f ical ly, the reaction mixturewas loaded onto a C,, Sep-Pak using 0.25 M tr iethylam-monium phosphate (TEAP), pH 3.5. The Sep-Pak wasr insed with 5 ml of H,O and the radiolabeled peptideeluted with TEAP /actonitri le (l/l). The radiolabeledpeptide was di luted in HZ0 and loaded onto the HPLCcontaining a C,, Bondapak resin. ( 51-Tyr0)NMB waseluted using a 0.1% TFA/GO% acetoni tri le gradient andthe speci f ic activ i ty was 2200 Ci/mm ol.

The I-NMB analogue was then used in receptorbinding studies. Sections of fresh frozen rat brain (20 pm


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NEIIROMEDIN B BINDIN(: SIIES

FIG. 3. Amount of J-NMH hound as a function of peptide con-centration. Let?: lhe amount of total (ai and nonspecific (A) I-NMBbound was determined as a function of radiolabeled peptide concentra-tion. The mean value t SE of three determinations is indicated. Thedifference between the two represents specific h indin g (0). Right: Scat-chard plot of the Qpecitic binding data.

(& = 1 nM) to a single class o f si tes (R,,;, , = 80 fmol/mgprotein).The speci f ic i ty of binding was investigated. Figure 4

shows that li t t le speci f ic binding was inhibited by 0.1 nMGRP whereas mos t speci f ic binding was inhibited by 1000nM GRP. The IC , , , for GRP was 60 nM. In cont rast NMBwas more potent wi th an IC,,, value of 4 nM whereasGRP was less potent wi th an IC,,, value of >lO,OOOnM. Also, GRP 7, ranatensin, l i tor in, and BN had I&,,values of 6, 20. 20, and 50 nM.

The distr ibution of %NMB binding si tes was inves-tigated using in vitro autoradiographic techniqu es and fi lmgrain concentrations were analyzed on the densi tometer.High grain densi t ies (5.9 fmol I-NMB/mg protein; Ta-ble 2) were present in t,he anter ior ol factory nucleus butnot control sections treated with 1 pM unlabeled NMB(Fig. 5A, 5G). Also, moderate grain densi t ies were presentin the external plexiform layer and internal granular layerof the ol factory bulb (Fig. 5A). High grain densi t ies werepresent in the ol factory tubercle whereas moderate graindensi t ies were present in the claustrum (Fig. 5B). Figure5C shows that high I-NMB grains were present in thenucleus accumbe ns (2.4 fmol/mg) whereas moderate graindensi t ies were present in the cingulate cortex and septalhippocampal nucleus (1.6 fmol/mg ). Moderate grain den-si t ies were present in the neocortex, especial ly layers Vand VI, whereas the densi t ies in layers I- IV were some-what lower (Fig. 5D). Also, moderate grain densi ties werepresent in the anter ior comm issure (Fig. 5D) and lowdensi t ies in the caudate p utamen (0.5 fmol/mg). Moderategrain densi t ies were also present in the bed nucleus of thestr ia terminal is (1.6 fmol/mg ), septal hypothalamic nu-cleus, suprachiasmatic nucleus, and medial preoptic areawhereas low grain densi t ies were present in the lateralpreoptic area and grains were absent in the corpus cal-losum (Fig. 5E). Low grain densi t ies were present in theglobus pallidus and subforn ical organ (Fig. 5E). High I-

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

NMB grains were present in the central medial thalamic(3.5 fmol/mg) nucleus, whereas moderate grain densi t ieswere present in the rhomboid and reunl lens thalamic nu-clei , paraventr icular hypothalamic nucleus (0.7 fmol/mg ),and central amygdaloid nucleus (1 .O f mol!mg). Moderate

TABLE 2I-NMH (irain Density in Rat Brain Regions

Brain region. coordinatesAnterior olfactory nucleus. 14.2 mmCentral medial thalamic nucleus. 7.2 mmNucleus accumbens. 10.2 mmSeptal hippocampal nucleus. IO.2 mmStria terminalis, 8.7 mmHippocampus. 7.2 mmSuperior olive, 0.3 mmCentral amygdaloid nucleus, 7.2 mmVentral medial hypothalamic nucleus, 5.7 mmMedial preoptic nucleus, 8.7 mmMedial amygdaloid nucleus, 5.7 mmDorsal parahrachia l nucleus. 0.3 mmParventricular hypothala mic nucleus. 7.2 mmLocus roeruleus, -0.3 mmEntorh inal cortex. 0.7 mmCaudate putamen. 9.2 mmCentral pray, 2.2 mmSubiculum, 4.2 mmNucleus t ractus solitarious. 3.X mmParie tal cortex. 8.7 mm

1)ensit.v11 nol/mg protein I

5.88 ~*- 0.29:~.?x i 0.89:!.-I:! + 0 . I!)1 .fi:! -t 0.25I .m 2 0. 131 4:1 I o.1:iI .oo t 0. IO

0.98 - 0. I2O.R9 + 0.060.81 I 0.070.80 t 0.1 I0.70 l 0.050.68 i 0.060.67 f 0.09o.r,:! i- 0.170.48 2 0.06O.l(i + 0.100. 11 i 0.090. 1 1 OF 0.070.38 7 0.06

Note. The autoradiog raphic films were compared to sl-microscalestandards on an Amersham RAS 3000 densitometer and the grain densitywas calculated. The nonspecific bind ing was subt ratted from the totalbinding to yield the specific binding data. The mean value + SE of sixdetermin ations is indicate d when the concentration of I-NMH wa>90 PM.


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164 LEE ET AL.

FIG. 5. Autoradiography of ?-NMB in the rat forebrain. The distribution of total *I-NMB binc g sites was determ ined using coronalslices derived from Pax inos and Watson (17) coordinates (A) 14.2 mm, (B) 11.2 mm, (C) 10.2 mm, (D) 9.2 m, (E) 8.7 mm, and (F) 7.2 mm. Also,nonspecific bindi ng was determi ned using slices derived from coordinates (G) 14.2 mm. Abbreviatio ns sed: anterior olfactory nucleus (AO),claustrum (Cl), olfactory tubercle (Tu), septohip pocampa l nucleus (SHi), nucleus accumbens (Acb), cingula te cortex (ACg), anterior commisure(ac), neocortex (Neo), parietal cortex (FrPa), septal hypothala mic area (SHY), bed nucleus of the stria terminal is (BST), me dial preoptic (MPO)hippocam pus (Hi), dentate gyrus (DG), central media l thalam ic nucleus (CM), rhombo id thalam ic nucleus (Rh), paraventricular nucleus of thehypothalam us (Pa) and central amygd aloid nucleus (Ce).

grain densities were present in the polymorphic layer of ventral medial hypothalamic nucleus (0.9 fmol 1251-NMBthe dentate gyrus as well as the oriens layer, stratum ra- bound/mg protein) and medial amygdaloid nucleus. Figurediatum, and the molecular layer of the hippocampus (1.4 6B shows that low grain densities were present in thefmol/mg) whereas grains were absent from the lateral subiculum (0.4 fmol/mg) and parietal cortex (0.4 fmol/thalamus. mg). Moderate and low lz51-NMB grains were present inHigh grain densities were present in the central medial the pontine reticular nucleus (Fig. 6C) and central graythalamic nucleus (Fig. 6A) and moderate densities in the (0.5 fmol/mg), respectively. Also, low grain densities were


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NEIJROMEDIN B BINDIN(: SITES 165

FIG. 6. Region al distr ihution of I-NMB bindin g sites. I-NMB autoradiography was determined using coronal s lices derived from Paxinosand 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 med ial thalamic nucleus (CM), med ial amygda loid nucleus (Me), ventromedial hypothalamus (VMH), suhirulum (S), parietal cortex(FrPa), central gray (CG), pontine nucleus (Pn), dorsal raphe (DR), entorhinal cortex (Ent), inferior coll iculus (IC). locus (w>rIIIeus (LX), dorsalparahrachial nucleus (DPB), superior olive (SO), nucleus o f the solitary tract (Sol). and cerebellum (Cer).

present in the substantia nigra reticular, ventral tegmentalarea, medial geniculate nucleus, and superior coll iculus(Fig. 6C). Moderate grain densities were present in thedorsal raphe (Fig. 6D) and low grain densities in the en-torhinal cortex (0.5 fmol/mg). Moderate grain densitieswere present in the superior olive (1.0 fmol/mg), locuscoeruleus (0.7 fmol/mg), and dorsal parabrachial nucleus(0.7 fmol/mg) whereas low grain densi ties were presentin the inferior colliculus and grains were absent from thepontine reticular nucleus (Fig. 6E). Low grain densitieswere present in the nucleus of the solitary tract (0.4 fmol/mg) whereas grains were absent from the parvocellularreticular nucleus and the cerebellum (Fig. 6F).

DISCUSSION

For several classes of peptides, such as CCK andopioids, there are multiple classes of receptors (18, 19).For the BN family of peptides it was recently proposedthat 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 highaffinity and NMB with low affinity (21 24). The NMBreceptor which is present on esophageal muscu laris mu-cosa and gastric smooth muscle cells binds NMB withhigh affinity and GRP with low affinity (25). Previously,the binding properties of (I-Tyr4)BN to rat brain weredetermined (26). Here we investigated the binding prop-erties and autoradiographic localiza tion of I-NMB tothe rat brain.

Using coronal sections which contained the nucleus ac-cumbens, I-NMB bound with high affinity. Bind ing wastime dependent and 1-NMB bound with high affinity(& = 1 nM) to a single class of sites (B,,,, = 80 fmol/mgprotein). Binding of 12I-NMB was strongly inhibited byNMB, BN, GRP, ranatensin, and litorin i:Cno values of4, 50, 60, 20, and 20 nA4, respectively) but, not GRP Ii.Because NMB, BN, and GRP but not GRP-le bind withhigh affinity to the speci fic I-NMB binding sites, theC-terminal may be essential for high affinitv binding ac-


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166 LEE ET ALtivity. Previously, we found that the C-terminal octapep-tide of BN or GRP was essential for high affinity (lz51-Tyr*)BN binding activity (26).The I-NMB binding sites were discre tely d istributedin the rat brain. High grain densities of lz51-NMB werepresent in the anterior olfactory nucleus and central me-dial thalamic nucleus and moderate densities in the hip-pocampus and locus coeruleus. Because each of these brainregions has a high density of NMB, the iI-NMB bindingsites may be activated by endogenous NMB. Moderategrain densities were present in the stria terminalis , para-ventricular hypothalamic nucleus, and central as well asmedial amygdaloid nuclei. Because these areas have a highdensity of GRP but not NMB, these lz51-NMB bindingsites may be activated by endogenous GRP.

The distribution of I-NMB binding sites is surpris-ingly simila r to that of (251-Tyr4)BN. In particular, thedensity of (I-Tyr4)BN grains was high in the nucleusaccumbens where BN alters dopamine turnover (27), andhigh in the hypothalamus where direct injection of BNcauses hypothermia (28), satiety (9), and altered gastricacid secretion (29). Also, the density of (1-Tyr*)BNgrains is moderate in cortex where BN stimulates phos-phatidylinositol turnover (7), moderate in the hippocam-pus where BN is an excitatory agent (30), and low in thecentral grey where BN i s an analgesic (31). The biologicalactivi ties of NMB in the CNS have not been investigated.Previously, it was found that guinea pig pancreaticacini, pituitary, Swiss 3T3, and human glioblastoma celllines bind (Tyr*)BN (& = 4 nM) but not NMB (&= 100 nM) with high affinity. Prelim inary data (Lee etal., unpublished) indicate that certain hypothalamic nucleibind NMB with approximately lo-fold lower affinity thandoes the nucleus accumbens. It is possible that these re-gions contain a unique receptor subtype which prefersBN and GRP relative to NMB. Alternatively, there maybe more enzymes, such as aminopeptidases, which degradeNMB more readily than BN or GRP. In Swiss 3T3 andhuman glioblastoma cells, I-GRP is cross-linked to aglycoprotein of 75 kDa (32). Recently, the Swiss 3T3 GRPreceptor was cloned and found to comprise 384 aminoacids and 7 hydrophobic domains (33). Thus the GRPreceptor has a structure reminiscent of other G-protein-coupled receptors such as the substance K receptor (34).

In the superior olive, however, moderate densities ofI-NMB but not (iz51-Tyr4)BN grains are present. Cur-rently, we are investigating if NMB binds with higheraffinity than does BN or GRP in this brain region. It ispossible that in this brain region, similar to the esophagealmuscularis mucosa, there is a receptor subtype whichprefers NMB relative to BN or GRP. The structure ofthe NMB receptor remains to be determined.In summary, because 1251-NMB binds with high affinityin the rat CNS to distinct brain regions, NMB may func-tion as a regulatory peptide in the mammalian brain.

AC KN OWLED GMEN T SThe authors thank Rena Gets for helpf ul discussions. This research

is supported in part by NSF Grant BNS-8815133.

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NEIJROMEDIN R BINDING SITES 167bond analog ue w ith potent an d specif ic receptor antagonist activ ity .J Bid Chrm. 263: 505 6-5060.Paxinos, c l., and C. Watson (19861. The Rat Brain in Stereotax~(oordinates. 2nd ed. Academic Press, San Diego.Jensen, R. T., S. A. Wank, W. H. Rowley, S. Sato, S. and J. D.Gardner (198 91. Interactions of cholecystokinin with pancreaticacinar cells : A well s tudied mode l of a peripheral action of CCK.Trend.c Pharmacol. Sci. 10: 418-423.Haynes, L. (1989). Opioid receptors and s ignal transduction. TrmdsEhnrmacoC Scz. 9: 309-311.\on Schrenck. T.. I . Heinz-Erian, T. Moran, S. A. Mantey, .J. D.(Gardner. and R. T. Jensen (19891. Characterization of a neurom edinB-preferr ing receptor in esophagus muscle: Evidence for subtypeot homhesin receptors. Amer. J. Fhysiol. 256: G74imG75 8.Westendorf. J. M., and A. Schonbrunn (198). Bomh esin stimulatesprolactin and growth hormone release hy pituitary cells in culture.Endoc r inolug? 110: :352-359.Zachary, I.. and E. Rozengurt (198.51. High aff inity receptors forpeptides of the hombe sin family in Swiss 3T3 cells . Pro

autoradiografia localizacao nmb

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