neuropeptide y (npy) and peptide yy (pyy) receptors in rat brain

Cellular and Molecular Neurobiology, Vol. 10, No. 4, 1990

Neuropeptide Y (NPY) and Peptide YY (PYY) Receptors in Rat Brain

Tatsunobu Ohkubo, ~ Masami Niwa, ~'3 Kimihiro Yamashita, ~ Yasufumi Kataoka, t and Kazuto Shigematsu 2

Received March 13, 1990; accepted March 28, 1990

KEY WORDS: neuropeptide Y (NPY); peptide YY (PYY); NPY receptor; PYY receptor; quantitative receptor autoradiography; rat brain.

SUMMARY

1. Specific binding sites for neuropeptide Y (NPY) and peptide YY (PYY) were investigated in rat brain areas using quantitative receptor autoradiography with 125I-Bolton-Hunter NPY (125I-BH-NPY) and 125I-PYY, radioligands for PP-fold family peptides receptors.

2. There were no differences between localization of 125I-BH-NPY and 125I-PYY binding sites in the rat brain. High densities of the binding sites were present in the anterior olfactory nucleus, lateral septal nucleus, stratum radiatum of the hippocampus, posteromedial cortical amygdaloid nucleus, and area postrema.

3. In cold ligand-saturation experiments done in the presence of increasing concentrations of unlabeled NPY and PYY, 125I-BH-NPY and 125I-pYY binding to the stratum radiatum of the hippocampus, layer I of the somatosensory frontoparietal cortex, molecular layer of the cerebellum, and area postrema was single and of a high affinity. There was a significant difference between the affinities of 125I-BH-NPY ( K o = 0.96 nM) and 125I-PYY binding (Kd = 0.05 nM) to the molecular layer of the cerebellum. The binding of the two radioligands to the other areas examined had the same affinities.

4. When comparing the potency of unlabeled rat pancreatic polypeptide (rPP), a family peptide of NPY and PYY, to inhibit the binding to the areas examined, rPP displaced 125I-BH-NPY and 125I-PYY binding to the area postrema

1 Department of Pharmacology 2, Nagasaki University School of Medicine, 12-4 Sakamoto-machi, Nagasaki 852, Japan.

2Department of Pathology 2, Nagasaki University School of Medicine, 12-4 Sakamoto-machi, Nagasaki 852, Japan.

3 To whom correspondence should be addressed.

539

0272-4340/90/1200-0539506.00/0 ~ 1990 Plenum Publishing Corporation

540 Ohkubo, Niwa, Yamashita, Kataoka, and Shigematsu

more potently than it did the binding to the stratum radiatum of the hippocampus, layer I of the somatosensory frontoparietal cortex, and molecular layer of the cerebellum.

5. Thus, the quantitative receptor autoradiographic method with 125 I-BH- NPY and lzsI-PYY revealed differences in binding characteristics of specific NPY and PYY binding sites in different areas of the rat brain. The results provide further evidence for the existence of multiple NPY-PYY receptors in the central nervous system.

I N T R O D U C T I O N

Neuropeptide Y (NPY), a member of pancreatic polypeptide (PP)-fold family peptides, is composed of 36 amino acid sequences with a structural similarity to peptide YY (PYY) (Allen et al., 1987; Tatemoto et al. , 1982). There is a widespread distribution of NPY-like immunoreactivity containing neurons (NPY neurons) in the brain (Allen et al., 1983; Chronwall et al., 1985). NPY is thought to play the role of a neurotransmitter regulating blood pressure (Carter et al., 1984), feeding and drinking behavior (Clark et al., 1985; Levine and Morley, 1984; Morley et al., 1987), sexual activity (Clark et al., 1987), locomotor activity (Heilig et al., 1988), memory processing (Flood et al., 1987), gastric acid secretion (Humphreys et al., 1988), circadian rhythm (Albers and Ferris, 1984), and secretion of anterior pituitary hormones (Chabot et al., 1987; McDonald et al., 1985). Specific receptors for NPY have, to some extent, been elucidated using membrane binding assay (Chang et al., 1985; Saria et al. , 1985; Und6n et al., 1984) and the receptor autoradiographic method (H~irfstrand et al., 1986; Martel et al., 1986; Nakajima et al. , 1986; Saavedra and Cruciani, 1988; Torda et al., 1988).

PYY-like immunoreactivity containing neurons (PYY neurons) have also been detected in the brain (Broom6 et al., 1985; Ekman et al. , 1986). Although the distribution of PYY neurons differs from that of NPY neurons, recent studies revealed that 125I-Bolton-Hunter NPY (125I-BH-NPY) and IzsI-PYY specifically labeled the same binding protein, a candidate of physiologically active receptor protein in the porcine brain (Inui et al., 1989). This finding supports the proposal that NPY and PYY may exert functions through the same receptor sites (Chang et al., 1985; 1988; Inui et al. , 1988; Laburthe et al., 1986; Martel et al., 1986). However, as two subtypes, NPY-Y1 and -Y2 receptors, have been noted in the periphery and central nervous system (Colmers et al., 1987; Wahlestedt et al. , 1986), investigations on the receptors of NPY and PYY in the brain may aid in clarifying the mechanism underlying the multiplicity of physiological properties of the two peptides associated with receptor heterogeneity. In the present study, we examined the autoradiographic localizations and differences in the characteristics of specific 125I-BH-NPY and 125I-PYY binding sites in rat brain areas.

M A T E R I A L S A N D M E T H O D S

Adult Wistar Kyoto rats (300-350 g) were given water and standard chow (F-2, Funabashi Farm Co, Japan) ad libitum, and housed at 24°C, with lights on

NPY and PYY Receptors in Rat Brain 541

0600 to 1800 hr. These rats were decapitated between 1000 and 1200 hr. The brains were immediately frozen by immersion in isopentane at -30°C. Frozen, 16-#m-thick coronal sections were cut in a cryostat at -14°C, thaw-mounted onto gelatin-coated slides, and stored overnight under vacuum at 4°C.

NPY binding sites were labeled in vitro by incubation with 125I-BH-NPY (sp act, -81.4 TBq/mmol, New England Nuclear, Boston). Incubation with 125I-PYY (sp act, -81.4 TBq/mmol, New England Nuclear, Boston) was performed under the same condition. Sections were preincubated for 30 min at 22°C with 200 #M guanosine 5'-triphosphate (GTP) in 15 mM Tris-HC1 buffer, pH 7.4, containing 158 mM NaCI, 4.75 mM KC1, 1.27 mM CaC12, 1.42 mM MgC12, 11 mM glucose, and then washed for 5 min at 22°C in modified Krebs-Ringer buffer [137 mM NaCI, 2.68 mM KC1, 1.8 mM CaC12, 1.05 mM MgC12, 1.0 mg/ml glucose, 0.1% (w/v) ascorbic acid, 0.05% (w/v) bacitracin, buffered with 20 mM Hepes, pH 7.4] containing 1.0% (w/v) bovine serum albumin (BSA) (Und6n et al., 1984). Sections were incubated for 4 hr at 22°C with 125I-BH-NPY or 125I-PYY in modified Krebs-Ringer buffer containing 1.0% BSA. Nonspecific NPY and PYY bindings were determined in the presence of unlabeled 1.0/~M NPY (Peptide Institute Inc., Japan) and 1.0/~M PYY (Peptide Institute Inc., Japan), respectively. To determine the binding characteristics, we used unlabeled NPY, PYY, and rat pancreatic polypeptide (rPP) (Peninsula Laboratories, Inc., USA) as displacers for IaSI-BH-NPY and 125I-PYY binding. After incubation, the slides were washed three times (10sec each) with ice-cold modified Krebs-Ringer buffer containing 1.0% BSA, rinsed quickly in ice-cold distilled water, and dried under a cold stream of air. Sections were placed in cassettes with ~25I-standards (125I-microscales, Amersham) and opposed to 3H-Ultrofilm (LKB Industries, USA). The films were exposed for 1 week at room temperature and developed in Kodak D19 developer for 5 min at 4°C. The optical densities of the Ultrofilm were measured by computerized microdensitometry (UHG 101, Unique Medical Co., Japan) and related to the concentration of radioactivity, as based on a comparison with standard curves generated by processing sets of 125I-standards with each autoradiogram (Israel et al., 1984).

Binding parameters, dissociation constant (Ko), and maximum binding capacity (Bmax) were calculated from the data of cold ligand-saturation experiments, using a LIGAND computer program (Munson and Rodbard, 1980). The data were expressed as means + SE. Differences in the binding parameters were assessed by one-way analysis of variance (ANOVA), using F test.

RESULTS

Localization of ~SI-BH-NPY and ~-~I-PYY Binding Sites in Rat Brain Areas

In the initial binding experiments done at 22°C with 157 pM 125I-BH-NPY, we found that specific binding to the stratum radiatum of the hippocampus, area postrema, and layer I of the somatosensory frontoparietal cortex reached a maximum at 3 hr, and a plateau was maintained for up to 5 hr (Fig. 1). Hence, the binding experiments were carried out at 22°C for 4 hr.


12

E " ~ 10 0

~ 8 Z

o m 6

Z i 4

Z II1 I f2

0 0

F i g . 1 .

, /

I I I I

1 2 3 4 5

TIME (hour) Effects of incubation time on specific binding of

125I-BH-NPY (157pM) to rat brain areas. Each point represents the mean of four determinations. 0, Stratum radiatum of the hippocampus; (3, area postrema; A, layer I of the somatosensory frontoparietal cortex.

Various brain regions were selectively labeled by 125I-BH-NPY and 125I-pYY (Fig. 2). The apparent concentrations of specific binding sites calculated from the total binding and nonspecific binding amounts obtained at the incubation with 55pM 125I-BH-NPY and lzSI-PYY are shown in Tables I, II and III. The concentrations of 125I-PYY binding sites were consistently higher than those of the ~25I-BH-NPY binding sites. Autoradiographic localizations of ~25I-pYY binding sites were comparable to those of 125I-BH-NPY binding sites.

Moderate densities of the binding of ~25I-BH-NPY and125I-PYY were observed in layers I-II of the frontparietal cortex and striate cortex. Layers III-VI had low densities of the binding. High densities were found in the primary olfactory cortex and claustrum.

In the olfactory areas, high densities were apparent over various regions. Moderate to high densities were present in the external plexiform layer and internal granular layer of the olfactory bulb. The binding to the anterior olfactory nucleus and tenia tecta was intense. Low densities of the binding were detected in the caudate putamen and globus pallidus. Interestingly, in septal and hippocampal regions, the highest level of binding densities was observed in the lateral septal nucleus, oriens layer, and stratum radiatum of the hippocampus and, also, subiculum and entorhinal cortex of hippocampal subfields. Various nuclei of the amygdala showed moderate to high binding densities. Higher densities were observed in the central nucleus, medial nucleus and stria terminalis. The anterior cortical nucleus manifested a high density of the binding.

In the thalamus, moderate densities of binding were found in the habenular nuclei, central medial nucleus, rhomboid nucleus, and reuniens nucleus. A moderate to high density was detected in the medial geniculate nucleus. The

NPY and PYY Receptors in Rat Brain

12~I-BH-NPY ~ I - P Y Y a ........

b

~2~I-BH-NP Y ~251-pyy

543

Fig. 2. Receptor autoradigraphic localization of specific 125 125 I-BH-NPY and I-PYY binding sites in rat brain areas. Consecutive, 16-/zm-thick tissue sections were labeled with 55pM 125I-BH-NPY and 125I-PYY in vitro. Autoradiograms were generated by apposition of labeled tissue sections to 3H-Ultrofilm for 4 days. A comparison in

125 the localization of I-BH-NPY binding sites (right side of 125T each column) was made with that of I-PYY binding sites

(left side). Abbreviations used are listed in Tables I, II, and III.

anterior area, suprachiasmatic nucleus, supraoptic nucleus, ventromedial nucleus, zona incerta, and supramamillary nucleus of the hypothalamus contained moderate to high binding densities.

The binding sites were discretely distributed with moderate densities over the olivary pretectal nucleus, compact part of the substantia nigra, central gray, superficial layers of the superior colliculus, and inferior colliculus. However, the reticular part of the substantia nigra was not labeled by the radioligands. Low densities were detected in the rostal linear nucleus raphe, dorsal raphe nucleus, and median raphe nucleus.


Table I. Quantitative Determination of 125I-BH-NPY and 125I-PPY Binding Sites in the Rat Brain

Apparent binding concentration (fmol/mg)

Brain areas lzSI-BH-NPY 125I-pYY

Cerebral cortex Frontal cortex, layers I-III (Fr) Primary olfactory cortex (PO) Frontoparietal cortex,

motor, layer I (M) Frontoparietal cortex,

somatosensory layer I (SS) Cingulate cortex, anterior (ACg) Cingulate cortex, posterior (PCg) Striate cortex, area 18, layer I (Str) Temporal cortex, auditory area,

layer I (Te) Claustrum (CI)

Olfactory areas Olfactory nerve layer (ON) Glomerular layer (GI) External plexiform layer (EPI) Internal granular layer (IGr) Anterior olfactory nucleus (AO) Tenia tecta (qT)

Corpus striatum Accumbens nucleus (Acb) Caudate putamen (CPu) Fundus striati (FS) Globus pallius (GP) Ventral pallidum (VP) Olfactory tubercle (Tu)

Septum and hippocampal regions Lateral septal nucleus (LS) Medial septal nucleus (MS) Oriens layer hippocampus (Or) Stratum radiatum hippocampus (Rad) Molecular layer dentate gyrus (mol) Entorhinal cortex (Ent) Subiculum (S)

1.44 3.41 2.02 10.65

1.09 2.55

1.04 2.51 0.82 1.93 0.54 1.16 1.52 3.58

1.09 2.85 1.88 6.10

0.54 2.61 0.54 4.99 1.09 13.63 1.68 15.72 3.01 15.32 2.99 13.89

0.71 3.10 0.51 1.05 0.59 2.71 0 0.34 0.73 2.90 1.00 4.83

2.78 10.36 1.43 4.64 1.82 10.44 2.58 13.73 1.00 8.62 2.37 11.48 2.18 10.20

a Consecutive tissue sections were incubated with 55 pM 125I-BH-NPY and 125I-pYY. Optical density measurements were converted into fmol/mg. Values are expressed as means of three rats.

Modera te to high densities of the binding were localized in several brain s tem nuclei. M o d e r a t e densities were observed in the dorsal parabrachia l nucleus and K611iker-Fuse nucleus. The locus coeruleus, m o t o r t r igeminal nucleus, and facial nucleus showed a low level of binding. M o d e r a t e grain densities were found in the medial vest ibular nucleus and nucleus of the solitary tract. A considerable amoun t o f 125I-BH-NPY and 125I-pYY binding was observed in the area post rema. The molecular layer of the cerebel lum had low binding densities.

Thus , the highest densities were observed in the anter ior o lefac tory nucleus, lateral septal nucleus, s t ra tum rad ia tum of the h ippocampus , pos te romedia l cortical amygdaloid nucleus, and area pos t rema at the incubat ion with 55 pM of the radioligands. However , the receptor numbers in the brain areas remain


Table 1I. Quantitative Determination of 125I-BH-NPY and 125I-pYY Binding Sites in the Rat Brain a


Brain area 125I-BH-NPY 125I-PYY

Amygdala Central nucleus (Ce) Lateral nucleus (La) Basolateral nucleus (BL) Medial nucleus (Me) Anterior cortical nucleus (AC) Posteromedial cortical nucleus (PMC) Stria terminalis (st) Bed nucleus stria terminalis (BST)

Thalamus Anteior nuclei (A) Habenular nuclei (Hb) Paratenial nucleus (PT) Paraventricular nucleus (PV) Central medial nucleus (CM) Rhomboid nucleus (Ph) Reuniens nucleus (Re) Lateral posterior nucleus (LP) Medial geniculate nucleus (MG) Dorsal lateral geniculate nucleus (DLG)

Hypothalamus Anterior area (AH) Lateral area (LH) Medial preoptic area (MPO) Suprachiasmatic nucleus (Sch) Supraoptic nucleus (SO) Periventricular nucleus (Pe) Arcuate nucleus (Arc) Ventromedial nucleus (VMH) Zona incerta (ZI) Supramamillary nucleus (SUM) Medial mamillary nucleus (MM) Posterior mamillary nucleus (NP)

2.21 8.51 1.18 4.64 1.09 3.93 2.61 8.97 1.95 8.55 3.68 14.95 2.19 11.20 0.54 2.09

0 1.28 1.09 5.86 0.54 5.80 0.54 2.03 1.46 4.83 1.61 4.45 1.73 4.35 0.67 2.32 1.50 7.37 0.54 1.16

1.19 9.18 1.12 6.46 0.54 3.67 1.41 9.15 1.51 7.53 0.73 2.47 1.18 5.35 1.59 7.25 1.55 7.44 1.64 8.70 0.91 3.68 0.54 3.29

"See Table I, footnote a.

speculative until data on the binding characterist ics of the radiol igands are obtained.

Characterization of ~SI-BH-NPY and ~SI-pYY Binding Sites

125I-BH-NPY and 125I-PYY binding sites in layer I of the somatosensory frontoparietal cortex, s t ra tum rad ia tum of the h ippocampus , area pos t rema, and molecular layer of the cerebel lum were character ized, using the cold ligand- saturat ion method . 125I-BH-NPY binding was displaced by unlabeled N P Y with a high affinity, and Scatchard analysis gave a straight line, the reby indicating that N P Y bound to a single popu la t ion (Fig. 3) A m o n g areas character ized in this study, the largest value of dissociation constant (Kd), that is, the lowest affinity, was observed in layer I of the somatosensory f rontopar ie ta l cortex. In the case of lzsI-PYY binding, a single popula t ion of the binding sites was also observed (Fig. 4). Of part icular interest was the observa t ion that the Kd value of I25I-PYY


Table ili. Quantitative Determination of 125I-BH-NPY and 125I-PYY Binding Sites in the Rat Brain a


Brain areas 124I-BH-NPY 125I-PYY

Midbrain Olivary pretectal nucleus (OPT) Ventral tegmental area (VT) Substantial nigra, reticular (SNR) Substantia nigra, compact (SNC) Superior eolliculus, superficial layers (SC) Central Gray (CG) Rostral linear nucleus raphe (RLi) Dorsal raphe nucleus (DR) Median raphe nucleus (MnR) Inferior colliculus (IC)

Pons and medulla Pontine nuclei (Pn) Locus coeruleus (LC) Dorsal parabrachial nucleus (DPB) K61liker-Fuse nucleus (KF) Motor trigeminal nucleus (MT) Facial nucleus (Fa) Raphe magunus nucleus (RMg) Medial vestibular nucleus (MV) Parvocellular reticular nucleus (PC) Lateral reticular nucleus (LR) Inferior olive (IO) Nucleus of solitary tract (Sol) Reticular nucleus medulla, dorsal (MdD) Reticular nucleus medulla, ventral (MdV)

Circumventricular organs Subfornical organ (SFO) Median eminence (ME) Area postrema (AP)

Cerebellum Molecular layer (Mo) Purkinje layer (Pu) Granular layer (Gr)

1.22 7.27 0.54 4.06 0 0 1.09 4.83 1.09 4.26 1.45 6.19 0.54 5.16 0.54 5.61 0.54 6.67 1.37 3.48

0.09 1.16 0.50 2.15 1.46 7.73 1.09 6.44 0.54 3.58 0.54 2.32 0.54 2.32 1.00 7.12 0.54 4.35 0.54 4.25 0.45 3.48 1.06 6.19 1.09 3.29 0.55 2.32

0.54 6.09 0.91 2.51 2.09 14.82

0.55 4.06 0 1.53 0 1.16

a See Table I, footnote a.

binding to the molecular layer of the cerebel lum was of the highest affinity (Table IV). A comparison of the binding affinities of the two radioligands showed that the Ko values of 125I-PYY binding sites were smaller than those of 125I-BH-NPY binding sites in all areas examined. We calculated the ratio of Ka's of 125I-BH-NPY binding to IESI-PYY binding. We noted a large value, 18.3 in the molecular layer of the cerebellum, when compared with the values obtained f rom the binding sites in the s tratum radia tum of the h ippocampus (1.61), area postrema (1.30), and layer I of the somatosensory frontoparietal cortex (1.19).

We compared the potency of unlabeled NPY, PYY, and rPP to displace specific 125I-BH-NPY and IESI-PYY binding (Figs. 2 and 3, Table V). PYY inhibited 125I-BH-NPY binding with a high affinity. The potency of PYY in inhibiting ~25I-BH-NPY binding to the areas examined was higher than that of NPY, with the exception that the binding to layer I of the somatosensory frontoparietal cortex was equipotently displaced by NPY and PYY. NPY also


a b z ~+ 1BOl O0 ro~- o~.:%~-~-~-~-~.~ o , ~ ~ s ! . • 7 ~ ~^ 1 ,BOI O0.0~-O.~o~-~ "0 ~.'o ~ "~\a~,. ~ ~[

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-LOG PEPTIDE (M) BOUNO (pM) -LOG PEPTIDE (M) BOUND (pM}

c d ~1oo,.,.~o . . . . . . . . . . . . . . ~[ ~1oo . . . . ~ . . . . . . . . . . . . . . o.4 I

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-LOG PEPTIDE (M) BOUND (pM) -LOG PEPTIDE (M) BOUND (pM)

Fig. 3. Cold ligand-saturation and displacement experiments of ~25I-BH-NPY binding to the stratum radiatum of the hippocampus (a), area postrema (b), layer I of the somatosensory frontoparietal cortex (c), and molecular layer of the cerebellum (d). A typical Scatchard plot (right panel in each figure) was obtained by displacing the binding of 42 pM t25I-BH-NPY by unlabeled NPY (O) using the LIGAND computer program. The rank order inhibition of the binding was PYY (0) > NPY (~) >>> rPP (A).

a b ~100 ~-" °+e:•'3"~- t'-z~ - z~'z~--zx'z~ " a \ e "~ 14 ~3~?o ~.~"~ 100 [r e>e:e~z~-z~-a-~..ON, k e nk 1412 ~'o [ o

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-LOG PEPTIDE (M) BOUND (pM) -LOG PEPTIDE (M) BOUND (pM)

c d ~0011 o~.o:e~3~-a-~-a-a~.~.e Ka 4 ~,~ ~ 100 r+ ~ - ~ - a - ~ - ~ - ~ - a . ~ 6 [

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-LOG PEPTIDE (pM) BOUND (pM) -LOG PEPTIDE (M) BOUND (pM)

Fig. 4. Cold ligand-saturation and displacement experiments of 125I-PYY binding to the stratum radiatum of the hippocampus (a), area postrema (b), layer I of the somatosensory frontoparietal cortex (c), and molecular layer of the cerebellum (d). A typical Scatchard plot (right panel in each

. . . . 125 figure) was obtained by displacing the binding of 42 pM I-PYY by unlabeled PYY (O) using the LIGAND computer program. The rank order inhibition of the binding was PYY (O)> NPY (0) >>> rPP (A).


Table IV. Binding Parameters, Dissociation Constant (Ka), and Maximum Binding Capacity (Bmax) of Specific mSI-BH-NPY and lzSI-PYY Binding Sites in the Rat Brain a

125I-BH-NPY binding 125I-pYY binding '

Brain areas Ka (riM) Bma x (fmol/mg) Kd (riM) Bma x (fmol/mg) K d ratio*

Rad 1.21 + 0.11 a 122 + 9.3 0.75 ± 0.05 c 160 ± 10.4 1.61 AP 1.20 ± 0.11 b 98.3 + 7.3 0.76 ± 0.05 f 139 ± 7.1 1.30 SS 3.45 ± 0.29 ~ 118 + 12.3 2.89 ± 0.44 g 178 ± 24.3 1.19 Mol 0.96 ± 0.24 d 5.5 ± 1.2 0.05 d: 0.01 h 4.4 ± 0.3 18.3

a Values are expressed as means i SE of individual four rats. Rad, stratum radiatum of the hippocampus; AP, area postrema; SS, layer I of the somatosensory frontoparietal cortex; Mol, molecular layer of the cerebellum.

* Values are the ratio of Kd'S of mSI-BH-NPY binding to 125I-pYY binding. Differences in the data were assessed by one-way ANOVA using the F test. NPY binding; P <0.01 ( a < c, F = 37.82; b < c, F = 37.36; d < c, F = 31.47). PYY binding; P < 0.01 (e < g, F = 17.47; f < g, F = 17.33; h < g, F = 31.15; h < e, F = 119.26; h < f, F = 129.24).

Table V. Inhibition (IC5o, nM) of Specific 125I-BH-NPY and 125I-pYY Binding by Unlabeled NPY, PYY, and rPP a'*

125I-BH-NPY binding 125I-pYY binding

Brain areas PYY NPY rPP PYY NPY rPP

Rad 0.52 + 0.07 ~ 2.17 + 0.08 c 4,580 + 300 i 0.97 ± 0.03 a 3.06 + 0.52 c >10,000 AP 0.38 + 0.07 b 2.26 + 0.34 f 483 + 101 j 0.90 + 0.08 b 2.24 + 0.32 f 1,050 + 210 i SS 3.45 + 0.38 c 4.06 + 0.44 g 4,950 + 1000 k 3.16 + 0.20 c 7.91 -t- 0.90 g 3,720 + 330 i Mol 0.06 ± 0 .003 d 1.01 :t: 0 .19 h >10,000 0.12 + 0 .01 d 0.28 + 0.05 h >10,000

a IC50 is the concentration of peptides displacing 50% of specifically bound 125I-BH-NPY (42 pM) and mSI-pYY (42 pM). Values are expressed as means i SE of individual four rats. Differences in the data were assessed by one-way ANOVA using the F test. Rad, stratum radiatum of the hippocampus, AP, area postrema; SS, layer I of the somatosensory frontoparietal cortex; Mol, molecular layer of the cerebellum.

*125I-BH-NPH binding, P<0 .01 ( a < b , F=56 .08 ; d < a , F=39 .74 ; e < g , F=17 .99 ; h < e , F = 31.23; j < i, F = 172.93; a < e, F = 225.20; e < i, F = 240.94; b < f, F = 20.88; f < j, F = 22.86; g < k , F=24.42; d < h , F=25.44) . 125I-PYY binding; P < 0 . 0 1 ( a < c , F = l 1 5 . 0 1 ; d < a , F = 605.54; e < g , F=21 .57 ; h < e , F=19 .98 ; i < j , F=38 .38 ; a < e , F=15 .87 ; b < f , F=22 .62 ; f < i , F = 25.00; c < g , F = 26.88; g < j , F = 123.38); P <0.05 ( d < h , F = 14.50).

i n h i b i t e d 125I-PYY b i n d i n g w i t h a h i g h a f f in i ty , h o w e v e r , t h e p o t e n c y o f N P Y w a s

s i gn i f i c an t l y w e a k e r t h a n t h a t o f P Y Y in al l f o u r r e g i o n s . A l t h o u g h r P P w a s a l so

a b l e t o d i s p l a c e 1 2 5 I - B H - N P Y a n d 125I-PYY b i n d i n g , r P P w a s f a r l ess p o t e n t t h a n

N P Y a n d P Y Y . I t w a s n o t e w o r t h y t h a t a m o n g t h e f o u r r e g i o n s , r P P d i s p l a c e d

1 2 5 I - B H - N P Y a n d 12~I-PYY b i n d i n g t o t h e a r e a p o s t r e m a m o r e p o t e n t l y t h a n i t

d i d t h e b i n d i n g to t h e o t h e r a r e a s .

D I S C U S S I O N

A u t o r a d i o g r a p h i c l o c a l i z a t i o n o f t h e 1 2 5 I - B H - N P Y b i n d i n g s i t e s w a s l a r g e l y

c o n s i s t e n t w i t h t h e e v i d e n c e r e p o r t e d b y H ~ i r f s t r a n d et al. ( 1 9 8 6 ) a n d M a r t e l et aL (1986) . W h e n t h e l o c a l i z a t i o n o f 1 2 5 I - B H - N P Y b i n d i n g s i t e s p r e s e n t e d h e r e is


compared with the immunohistochemical distribution of NPY neurons reported elsewhere (Chronwall et al., 1985; De Quidt and Emson, 1986), we noted an anatomical "receptor-transmitter mismatch." A considerable amount of 125I-BH- NPY binding was found in brain areas such as the compact part of substantia nigra, superior colliculus, median raphe nucleus, inferior colliculus, motor trigemi- hal nucleus, raphe magnus nucleus, medial vestibular nucleus, inferior olive, subfornical organ, area postrema, and molecular layer of the cerebellum, areas with no evidence of NPY neurons. On the contrary, no 125I-BH-NPY binding sites were detected in the corpus callosum, an area rich in NPY neurons. Distribution of NPY neurons in the anterior olfactory nucleus and various hippocampal subfields was scanty but in these areas the highest amounts of ~25I-BH-NPY binding sites were found. The same phenomenon was first noted in the anatomical correlation between substance P receptor and substance P immunoreactivity-containing neurons (Mantyh et al. , 1984). While the reason for mismatch remains unclear (Kuhar, 1985), according to the hypothesis of Shultzberg and H6kfelt (1986), a "quiet" moiety of the receptor may be present, or the endogenous ligand reaching via diffusion over a long distance may activate the receptor.

The distribution of 125I-PYY binding sites corresponded well with that of the 125I-BH-NPY binding sites, although immunohistochemical studies revealed the PYY neurons to be discretely localized only in the lower brain stem, spinal cord, and hypothalamic areas (Broom6 et al. , 1985; Ekman et al., 1986). There are no differences between the biological activities of the NPY and PYY injected into the paraventricular nucleus, an area where no PYY neurons innervate, as far as increasing carbohydrate consumption is concerned (Stanley et al. , 1985). Hence, as PYY neurons are far less abundant when compared with NPY neurons, 125I-PYY seems to be a radioligand recognizing NPY receptors in the most parts of the rat brain. In fact, the affinity cross-linking technique recently revealed that ~25I-PYY and ~2SI-BH-NPY labeled the same single band with a molecular mass of 50,000 daltons, a finding supporting the proposal that NPY and PYY share a common receptor in the porcine brain (Inui et al., 1989). Laburthe et al. (1986) reported evidence suggesting the possible existence of a common NPY-PYY receptor in the rat small intestinal epithelial cells, defined as "PYY-preferring receptor site," which has a high affinity for PYY than NPY. The present data that 125I-PYY bound to rat brain areas with a high affinity than did ~25I-BH-NPY and unlabeled PYY displaced both radioligands binding more potently than did NPY strongly support the existence of the same receptor as PYY-preferring receptor site in the rat brain. NPY and PYY, PP-fold family peptides, with a high degree (70%) of sequence homology are produced by a distinct gene and gene-encoding processes (Higuchi et al., 1988). Similarly, among the family peptides studied, atrial natriuretic peptides and recently discovered brain natriuretic peptides, natriuretic family peptides, produced separately seem also to activate the common receptor to exert biological functions (Chang et al. , 1989; Maeda et al., 1989, 1990).

Multiple subtypes of NPY receptors have been postulated (Schwartz et al., 1987; Walker and Miller, 1988). In the sympathetic nervous system, it seems


likely that there are two subtypes, NPY-Y1 and -Yz receptors (Wahlestedt et al. , 1986). The NPY-Y2 receptor locates prejunctionally on the sympathetic nerves, and the NPY-Y1 type presumably exists on the postsynaptic sites. The NPY-Y2 receptor originally characterized in peripheral areas was also found to locate in the hippocampus (Colmers et al. , 1987). Sheikh et al. (1989) obtained evidence for characteristics of the two types of NPY receptors in the nervous system, using monoiodinated radioligands of a long C-terminal fragment, NPY (13-36), and NPY. The NPY-Y1 receptor has a Kd value in a nanomolar range, and the Kd of the NPY-Yz receptor was calculated to have a subnanomolar value. The finding of the existence of low- and high-affinity sites in the brain seems pertinent to the present observations that 125I-BH-NPY and 125I-PYY bound to the molecular layer of the cerebellum with subnanomolar Ko'S , whereas the radioligands bound to the layer I of the somatosensory frontoparietal cortex with Kd'S of 3.45 and 2.89 nM, respectively. According to the hypothesis of Sheikh et al. (1989), the binding sites in the layer I of the somatosensory frontoparietal cortex should be a subtype of the NPY-Y1 receptor. Furthermore, the difference between the affinity of 125I-BH-NPY and lzSI-PYY binding sites is particularly noteworthy. As indicated by the ratio of Kd'S of 125I-BH-NPY and 125I-pYY binding (molecular layer of the crerbellum, 18.3; stratum radiatum of the hippocampus, 1.61; area postrema, 1.30; layer I of the somatosensory frontoparietal cortex, 1.19), the binding sites in the molecular layer of the cerebellum were preferentially labeled by 125I-PYY with a much higher affinity than by 125I-BH-NPY. This may be evidence suggesting the existence of a further heterogeneity of the receptors in the brain. Thus, comparison of the binding affinities of the two radioligands, 125I-PYY and lzSI-BH-NPY, paves the way for studies on the heterogeneity of brain NPY-PYY receptors.

In summary, we localized and characterized ~zSI-BH-NPY and ~25I-PYY binding sites in the rat brain using the quantitative receptor autoradiographic method. As we observed the difference between the binding characteristics of 125I-BH-NPY and ~25I-PYY in rat brain areas studied, the possible existence of multiple receptors for NPY and PYY, PP-fold peptides, in the central nervous system warrants further investigations.

ACKNOWLEDGMENTS

We thank Professor Emeritus Masayori Ozaki for support, M. Ohara for pertinent comments, and M. Hirata for secretarial services.

NOTE A D D E D IN PROOF

While our manuscript was in preparation, Lynch et al. (1989) provided evidence suggesting the existence of NPY receptor subtypes in the rat brain, based on the data of membrane binding and receptor autoradiographic methods with monoiodinated radioligands of NPY and PYY.


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