BRAIN RESEARCH

ELSEVIER Brain Research 705 (1995) 177-187

Research report

Expression of a novel angiotensin II receptor subtype in gerbil brain

Ana Maria de Oliveira, Mohan Viswanathan, Frank M.J. Heemskerk, Juan M. Saavedra *

Section on Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, 10 Center Drive MSC 1514, Bldg 10, Room 2D-45, Bethesda, MD 20892-1514, USA

Accepted 29 August 1995

Abstract

Angiotensin II receptors are highly localized in adult gerbil brain. Apparent receptor number is high in subfornical organ, vascular organ of the lamina terminalis, nucleus of the solitary tract, hippocampus, and in the anterior pituitary gland. In the hippocampus, binding is localized to the stratum oriens, radiatum, the lacunar molecular layers of the CA1 subfield, and the molecular layer of the gyrus dentatus, with a medial to lateral and anterior to posterior gradient in receptor expression. Binding is absent from the pyramidal layer of the CA1 subfield and from the granular cell layer of the gyms dentatus, areas rich in angiotensin IV binding. Characterization in the hippocampus revealed the presence of a high affinity receptor, sensitive to incubation with the guanine nucleotide GTPTS, and displaced by angiotensin II = angiotensin III < Sarl-IleS-angiotensin II, but not by angiotensin IV or other angiotensin fragments, the AT 1 receptor antagonist losartan, or the AT 2 ligands CGP 42112 or PD 123177. In other brain areas, binding was equally insensitive to displacement by AT 1 or AT 2 ligands, with the exception of binding in the olfactory bulb, which was totally displaced by CGP 42112 and PD 123177, but not by losartan. In the gerbil, most of the brain and pituitary angiotensin II receptors are different from the AT 1, AT 2 and AT 4 subtypes, and should be considered 'atypical' until further characterization.

Keywords: Hippocampus; Circumventricular organ; Guanine nucleotide; Brain angiotensin system; Receptor pharmacology; Quantitative autoradiography

1. Introduct ion

The existence of a brain angiotensin II (Ang II) system in mammals is well established [14,22]. Stimulation of brain Ang II receptors regulates the well known central effects of Ang II, the modulation of drinking, salt appetite, and autonomic and pituitary function [14].

The Ang II receptor heterogeneity, first described in the periphery [1,19] applies also to the brain [14,20]. There are two major receptor subtypes, AT 1 and AT 2. Both AT 1 and AT 2 receptors have been cloned, revealing the presence of seven transmembrane domains characteristics of G-protein linked receptors [8]. The AT 1 receptors, antagonized by selective ligands such as losartan, seem to mediate most, if not all, classical central actions of Ang II [1,8,19]. The function of the AT 2 receptors, on the other hand, has not yet been clarified [1,19,21].

* Corresponding author. Fax: (1) (301) 402-0337.

0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)01158-7

Further studies revealed additional heterogeneity for central Ang II receptors. In the rat, two classes of AT 1 receptors, the ATIA and the ATIB subtypes, have been distinguished based on their cDNA sequences, and have been found differentially localized and regulated [8]. Two kinds of AT 2 receptors may also coexist in brain, some probably linked (AT2A) and some not linked (AT2B) to G-proteins [21].

In addition, binding sites for an Ang II fragment, an- giotensin(3-8) (Ang IV) (AT 4 binding sites) have been described in mammalian brain [18]. Thus, more complete studies reveal increased heterogeneity in brain Ang II receptors and related binding sites. The expression of the different receptor subtypes is dependent on the brain area studied, the developmental stage, and the mammalian species considered [14].

We now report, on the basis of the ligand affinity profile, the presence of still another Ang II receptor sub- type, in the gerbil brain and pituitary gland. This receptor has characteristics different from those of any of the Ang II receptors previously described in the mammalian brain.

178 A.M. de Oliueira et a l . / Brain Research 705 (1995) 177-187

2. Materials and methods

2.1. Animals

We used male Mongolian gerbils weighing 65-80 g, from Tumblebrook Farm, West Brookfield, MA. All ani- mal procedures were approved by the NIMH Animal Care Committee. The animals were kept under controlled condi- tions of light (12:12 h light/dark cycle, with lights on at 07.30 h) for one week, and were provided with food and water ad libitum. Animals were killed by decapitation between 09.00 and 10.00 h. The brains and pituitary glands were immediately removed, frozen at -30°C by immer- sion in isopentane on dry ice, and were kept frozen at

-70°C. For quantitative autoradiography, coronal brain sections, and sections of the pituitary gland, 16 /zm thick, were cut in a cryostat at -17°C, thaw mounted on gelatin-coated glass slides and dried overnight in a desicca- tor at 4°C [20].

2.2. Ligand binding

Ang II binding was determined by incubation of brain and pituitary sections with the Ang II agonist [125I]Sarl- Ang II [20]. Tissue sections were preincubated for 15 min in 10 mM phosphate buffer, pH 7.4, containing 120 mM NaC1, 5 mM MgC12, 0.005% bacitracin (Sigma Chemical Co., St. Louis, MO) and 0.2% proteinase-free bovine

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Fig. 1. Stability of [125I]Sarl-Ang II after incubation with gerbil brain sections. Analysis by HPLC of [125I]Sarl-Ang II in the incubation buffer after 2 hours of incubation in the absence (A) or the presence (B) of the protease inhibitor mixture, as described in Section 2. Standard [125I]Sarl-Angll elutes at 14.2 min in this system (indicated with an arrow)• Dotted line indicates acetonitrile gradient in 0.1% trifluoroacetic acid. Solid line indicates radioactivity

in eluted fractions•

A.M. de Oliveira et al. / Brain Research 705 (1995) 177-187 179

serum albumin, fol lowed by incubation for 120 min at 22°C in fresh buffer, prepared as above with addition of 50 /xM Plummer ' s inhibitor, 100 /zM phenylmethylsulfonyl fluoride (PMSF), 500 p~M phenanthrolin, and the appropri- ate l igand concentration. After incubation, the sections were washed four times for 1 min each, in fresh ice-cold 50 mM Tris-HCl buffer (pH 7.4), fol lowed by a 30 s wash in water at 0°C.

To localize brain Ang II receptors, we used coronal sections of the forebrain and brain stem. Sections were selected at areas where functional Ang II receptors have been previously localized by autoradiography in the rat [20]. Sections were incubated with 5 X 10-~° M [125I]Sara-Ang II, and non-specific binding characterized by incubation of consecutive sections with 10 -6 M unla- beled Ang II.

To characterize the Ang II receptor subtype, consecu-

tive brain coronal sections, and pituitary sections, were incubated with 5 X 10 10 M [a25I]Sarl-Ang II alone (total

binding), or in the presence of 10 -6 M unlabelled Ang II (non-specific binding), or 10 -6 M losartan, to displace binding to AT t receptors, or 10 6 M PD 123177 or 10 -6 M CGP 42112, to displace binding to AT 2 receptors [20], or 10 -6 M unlabeled Ang IV to displace binding to the A T 4 site [18].

Further Ang II receptor subtype characterization was completed in the hippocampus, using consecutive coronal sections cut at a level - 1.4 to - 1.7 mm caudal to bregma [10]. Saturation assays were performed in consecutive sections incubated in the presence of [ lesI]Sarl-Ang II, with concentrations between 1 x 10 -1~ to 1 × 10 -8 M. The competi t ion assays were conducted using 5 X 10 ~0 M [~25I]Sar1-Ang II, displaced by 10 -12 to 1 x 10 -6 M of

unlabeled Ang II, Ang III, Sarl-I leS-Ang II, the AT 1

A D

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( a ) , t 1.,., (b) Fig. 2. Localization of angiotensin II binding in gerbil brain, a: level of the solitary tract (A, B, C) and level of the subfornical organ (D, E, F). A and D, Toluidine blue staining. B and E, total binding, incubated in the presence of 5 X 10 -1° M [125I]SarX-Ang II. C and F, non-specific binding, incubated as above, with addition of 10 - 6 M unlabeled Ang II. b: level of the paraventricular nucleus. A, Toluidine blue staining; B, total binding; C, non-specific binding. NTS, nucleus of the solitary tract; IO, inferior olivary complex; SFO, subfornical organ; LT, lamina terminalis; OVLT, vascular organ of the lamina terminalis; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus.

180 A.M. de OliL,eira et al. / Brain Research 705 (1995) 177-187

antagonist losartan and the AT 2 competitor CGP 42112. In addition, sections incubated with 5 X 10 - l ° M [t25I]Sarl- Ang II were incubated with single concentrations (10 6 M) of several Ang II fragments (Ang 1-5, Ang 1-6, Ang 1 - 7 , terminal tripeptide, terminal tetrapeptide and Ang IV.

For studies on the localization of the AT 4 binding site, consecutive brain sections containing the hippocampus, at the level of - 1 . 4 to - 1 . 7 mm caudal to bregma, were incubated as described above, in the presence of 5 X 10 10 M [mSI]Tyr2-Ang IV [6]• Non-specific binding was deter- mined in the presence of 10 6 M unlabeled Ang IV [6]. Additional displacement experiments were conducted by incubating consecutive sections in the presence of 10 -6 M unlabeled Ang II, 10 -6 M losartan, 10 -6 M PD123177 or 10 -6 M CGP 42112.

The influence of guanine nucleotides on Ang II binding was analyzed by incubating consecutive hippocampal sec- tions with 5 X 10 -1° M [mSI]SarKAng II in the presence or absence of 10 9 M to 10 -4 M GTPTS or 10 -4 M

ATPyS [211.

2.3. Quantitative autoradiography

Dry sections were exposed to Hyperfilm-(3H) along • 1 2 5 •

with 16 /zm sections of I-labeled Mlcroscale standards. Films were developed in Kodak D-19 developer for 4 rain at 0°C, fixed in Kodak rapid fixer for 4 min at 22°C and rinsed in water for 10 rain. Optical densities were quanti- fied by computerized microdensitometry using the Image 1.42 program (NIMH, Bethesda, MD). The optical densi- ties were related to the concentration of radioactivity pre- sent in the sections by comparison with 125I standard

curves, as described [13]. All results were analyzed by non-linear regression using the InPlot System (GraphPad Software, version 4, 1992). Brain regions were identified by comparison with Toluidine blue staining of consecutive sections.

2.4. Analysis of ligand metabolism

The metabolism of [125I]SarJ-Ang II was analyzed by reverse phase high performance liquid chromatography (HPLC) using a linear gradient (15-40% acetonitrile in 0.1% TFA) over 12.5 min at 2 m l / m i n ) on a C-18 column (250 x 4.6 mm, 5/xm). In this system, intact [125I]Sarl-Ang

II eluted at 33.4% acetonitrile (tRT = 14.2 rain). To deter- mine possible ligand metabolism, aliquots of the incuba- tion buffer before and after incubation were analyzed by HPLC.

2.5. Materials

Sarl-Angiotensin II was purchased from Peninsula Lab- oratories, Belmont, CA, and was iodinated by NEN- DuPont, to a specific activity of 2200 C i /mmol . [lz5I]Tyr2-Ang IV was purchased from NEN-DuPont

(specific activity 2200 Ci /mmol ) . Ang II, Ang III, Sarl ,I leKAng II, Ang IV, and the Ang

II fragments Ang 1-5, Ang 1-6, Ang 1-7, Ang terminal tripeptide, Ang terminal tetrapeptide, were purchased from

Peninsula Laboratories. Losartan (DuP753, {2-n-butyl-4-chloro-5-hydroxy-

methyl- 1-[2'(1H-tetrazol-5-yl)biphenyl-methyl]imidazole} was a gift from DuPont-Merck, Wilmington, DE. PD

Table 1 Angiotensin II receptors in gerbil brain and pituitary gland

Specific binding Angiotensin II binding (fmol/mg protein)

After losartan After CGP 42112 After PD 123177

Atypical receptor: Subfornical organ 53 + 17 79 i 17 73 + 5 56 ± 12 Nucleus tractus solitarius 20_+ 1 25 ± 2 19 _+ 2 21 ± 1 Vascular organ of lamina terminalis 26 + 7 27 + 9 27 + 10 20 + 8 Laminaterminalis 7+ 3 8± 5 7+ 3 7± 4 Inferior olivary complex 7 + 1 10 ± 1 9 + 1 8 ± 0 Paraventricular nucleus 5 + 1 6 ± 1 5 + 1 5 ± 1

Hippocampus StratumoriensofCA1 14+ 1 10+ 1 8± 2 13+ 1 Stratum radiatumofCA1 23+ 5 19+ 5 17+ 4 21+ 5 Subiculum 6+ 1 8_+ 1 6± 1 6+_ l Stratum molecularis of gyrus dentatus 5 + I 5 + 1 8 _+ 1 6 _+ 2 Median eminence 1_+ 0 1_+ 0 1 + II l_+ 0 Anterior pituitary 4+ 0 3_+ 1 3+ 1 4_+ 1 AT 2 Receptor: Olfactory bulb 4 _+ 1 4 _+ 1 ND ND

Consecutive sections were incubated with 5 x 10 -5 M [125I]SarKAng II, with (non-specific binding) or without (total binding) 10 6 M Ang II, or with losartan, CGP 42112 or PD 123177 (10 6 M) as described in Section 2. The numbers are the means + S.E.M. from 3-6 different determinations. ND, not detectable above non-specific binding.

A

A.M. de Oliveira et al. /Bra in Research 705 (1995) 177-187

B C

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Fig. 3. Characterization of angiotensin II binding in gerbil olfactory bulb. A: Toluidine staining. B: total binding, incubated in the presence of 5 × 10- t 0 M [1251]Sarl-Ang II. C: non-specific binding, incubated as above, with addition of 10-6 M unlabeled Ang II. D: displacement with 10 6 M losartan. E: displacement with 10 6 M PD 123177. F: displacement with 10 -6 M CGP 42112. GI, glomerular layer; Mi, mitral cell layer; EPI, external plexiform layer.

123177, 1-(4-amino-3-methylphenyl)methyl-5-diphenyl- acetyl-4,5,6,7-tetrahydro-lH-imidazo[4,5-c]pyridine-6-car- boxylic acid-2HCl was a gift from Parke Davis, MI. CGP 42112 [nicotinic acid-Tyr-(N.~-benzyloxycarbonyl-Arg)-

Lys-His-Pro-Ile-OH] was purchased from Neosystems Laboratory, Strasbourg, France.

Bacitracin, proteinase-free bovine serum albumin, PMSF, phenanthrolin, GTPTS and ATPyS were purchased

PCL(CA1) SO(CA1)

SG(GD) SM(GD)

SR

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Fig. 4. Autoradiographic localization of angiotensin II binding in gerbil hippocampus, coronal section. Coronal section at the level - 1.7 caudal to hregma. Upper figure: Toluidine blue staining. Lower figure: total binding, incubated in the presence of 5 × 10-10 M [1251]Sar]-Ang II. PCL(CA1), pyramidal cell layer of the CA1 field; SO(CA1), stratum oriens of the CA1 field; S1;'., stratum radiatum; SG(GD), stratum granulosum (granular molecular layer) of the gyrus dentatus; SM(GD), stratum molecularis (lacunar molecular layer) of the gyrus dentatus; CA3, CA3 field; CA4, CA4 field.

182 A.M. de Oliceira et al. / Brain Research 705 (1995) 177-187

from Sigma Chemical Co., St. Louis, MO; DL-2- mercaptomethyl-3-guanyl-dinoethylthiopropanoic acid (Plummer's inhibitor) was purchased from Calbiochem, La

Jolla, CA. Hyperfilm-(3H) and ~25I-labeled Microscale standards were from Amersham Corporation, Arlington Heights, IL; D-19 developer and rapid fixer from Eastman

[~2~i] Sar ~ _ Ang ]1 H ISTOLOGY [~2sI] - Ang IV

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A.M. de Oliueira et al. /Brain Research 705 (1995) 177-187

5O

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Fig. 6. Saturation analysis by quantitative autoradiography of []2sl]Sarl- Ang II binding to gerbil hippocampus. Consecutive brain sections were incubated with increasing concentrations of [125I]Sarl -Ang II, as described in Section 2. Each point is the mean + S.E.M., of 4-5 animals, measured individually. Inset: Scatchard plot.

Kodak Co., Rochester, NY; acetonitrile from Baker, Phillipsburg, N J, and T F A from Pierce, Rockford, IL.

3 . R e s u l t s

3.1. Analysis o f ligand metabolism

In our previous studies of rat brain sections incubated with [125I]Sarl-Ang II in phosphate buffer containing a low concentration of bacitracin [20], there was no ligand degra- dation, as determined by HPLC of aliquots of incubation medium (unpublished results). However, when we incu- bated gerbil brain sections with [125I]Sarl-Ang II in the same buffer, we observed the formation of at least 2 radioactive breakdown products of [t25 i]Sarl_Ang II in the incubation medium (Fig. 1A). This degradation was pre- vented by the addition of PMSF, phenanthrolin and Plum- mer ' s reagent to the incubation buffer, as can be seen in

Fig. lB. The incubation buffer for all subsequent experi- ments was modified accordingly.

3.2. Binding localization

Highest numbers of [125I]Sarl-Ang II specific binding were located in the subfornical organ, fol lowed by the vascular organ of the lamina terminalis, the nucleus of the solitary tract, and the hippocampus (Fig. 2, and Table 1). Significant Ang II binding was also present in the lamina terminalis, which in the adult rat is occupied by cells of the median preoptic nucleus [17], the paraventricular nucleus, the olfactory bulb, the median eminence, and in the ante- rior pituitary gland (Table 1; and Fig. 2, Fig. 3). Con- versely, no Ang II binding was found in association with large cerebral arteries or in the suprachiasmatic nuclei (Fig. 2B).

In the gerbil hippocampus, Ang II binding presented a medial to lateral, anterior to posterior gradient (Figs. 4 and

Fig. 5. Comparative autoradiographic localization and characterization of angiotensin II and angiotensin IV binding in coronal sections of gerbil hippocampus. Top figure: Toluidine blue staining. Left panel: [1251]Sarl-Ang II binding. Arrows point to the subiculum. Right panel: [12Sl]Ang IV binding. Arrowheads point to the pyramidal layer. None, total binding, incubated in the presence of 5 × 10 - l° M [lzSI]Sart-Ang II (left figures) or 5 X 10 -l° M [1251]Ang IV (right figures). Ang II, consecutive section incubated as above, with addition of 10 - 6 M unlabeled Ang Ii. Losartan, consecutive section, incubated as in None, with addition of 10 - 6 M of losartan. CGP 4211.2, consecutive section incubated as in None, with addition of 10 - 6 M CGP 42112. Ang IV, consecutive section, incubated as in None, with addition of 10 - 6 M Ang IV. Sections are taken at the level of - 1.5 mm caudal to bregma [10].

184 A.M. de OliL,eira et al. / Brain Research 705 (1995) 177-187

5). Binding was localized in well defined layers, and restricted to the stratum oriens, stratum radiatum, the lacunar molecular layer of the CA1 subfield, and the molecular layer of the gyms dentatus (Fig. 4). Conversely, [~25I]SarLAng II binding was absent from the pyramidal layer of the CA1 subfield and from the granular cell layer of the gyrus dentatus (Fig. 4).

The localization of hippocampal Ang II binding con- trasted with that of Ang IV binding in this area. Within the hippocampus, Ang IV binding was concentrated to the pyramidal layer of the CA1 and CA3 subfields and to the granular cell layer of the gyrus dentatus, areas devoid of Ang II binding (Fig. 5). In addition to its hippocampal localization, [125I]Ang IV binding was widely distributed

to cortical and subcortical areas, and it was insensitive to incubation in the presence of unlabeled Ang II, losartan, or CGP 42112 (Fig. 5).

3.3. Characterization of angiotensin H binding

We first attempted to characterize the Ang II receptor subtype present in the gerbil brain and pituitary gland. In almost all areas studied (Fig. 5, Table 1), neither incuba- tion in the presence of losartan or CGP 42112 was able to significantly reduce the Ang II binding. Binding in the olfactory bulb was the exception (Fig. 3), since all specific binding, which was localized to the external plexiform layer, was displaced by incubation in the presence of CGP

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Fig. 7. Competition studies of [12sl]SarhAng II binding in gerbil hippocampus. Competition curves are means _+ S.E.M. obtained from consecutive sections from 4 - 5 gerbils measured individually and incubated in presence of 5 × 10-10 M [t25I]SarLAng II and increasing concentrations of competitors as described in Section 2. Results are expressed as % specific binding (after substraction of nonspecific binding, obtained by incubation in presence of 10 -6 M Ang ll). A: competition by Ang II. B: competition by Ang III. C: competition by Sarl,Ile8-Ang If. D: competition by losartan and CGP 42112.

A.M. de Oliveira et aL / Brain Research 705 (1995) 177-187 1 8 5

42112 or PD 123177 (Fig. 3). None of the brain areas studied, or the pituitary gland, exhibited any significant losartan-sensitive binding (Fig. 5 and Table 1).

Further characterization of Ang II binding was per- formed in hippocampal coronal sections. Saturation studies of [125I]Sara-Ang II binding revealed a high affinity ( K d 0.64 X 10 - 9 M ) single binding site, with an approximate Bma x of 36.4 + 3.2 f m o l / m g protein (Fig. 6).

Competition studies indicated that [a25I]Sarl-Ang II binding was totally inhibited by unlabeled Ang II (Fig. 7A), Ang III (Fig. 7B), and Sarl,IleS-Ang II (Fig. 7C). Conversely, losartan or CGP 42112 did not inhibit binding, up to a concentration of 1 x 10 -6 M (Fig. 7D). Analysis of displacement curves indicated that the data fit a two site model for Ang II displacement ( F = 10.4; P = 0.0111) with a high affinity site comprising about 20% of the sites ( K i 7.1 × 10 -12 M) and a lower affinity site comprising about 80% of the sites (K i 9.5 X 10 -9 M) . Analysis of Ang III displacement indicated a better, although not sig- nificantly different ( F = 4.6; P --- 0.0726) fit for a two site model with a high affinity site (30% of sites) with K i

8 X 10- ~ M, and a lower affinity site (70% of sites) with g i 1 .8 X 10 -8 M. In addition, [125I]Sarl-Ang II binding was not inhibited by Ang IV (Fig. 6), or by the Ang II fragments Ang 1-5, Ang 1-6, Ang 1-7, terminal tripep- tide, and terminal tetrapeptide (results not shown).

3.4. Effects of guanine nucleotides

Incubation of brain sections in the presence of increas- ing concentrations of the guanine nucleotide GTPTS re- sulted in a progressive loss of [125I]SarNAng II binding,

~00 ? o~ c

X 3 C-

25

m 50

i i L i i ~

-9 -7 -5 GTP~S [log M]

Fig. 8. Effect of GTPyS on gerbil hippocampal [125I]Sarl-Ang II binding. Consecutive coronal brain sections were incubated with 5× 10 -1° M [1251]Sarl-Ang II and increasing concentrations of GTPyS, as described in Section 2. Results are expressed as means + S.E.M., of groups of 4-5 rats measured individually.

with a K i of 3.8 × 10 -8 M (Fig. 8). Conversely, ATPyS, at 1 × 10 4 M, did not affect [lasI]Sarl-Ang II binding (results not shown).

4. Discussion

The brain Ang II system, including specific receptors to Ang II, plays important roles in all mammalian species [14]. However, early observations, performed with the use of radiolabeled Ang II in membrane preparations from large areas, suggested that Ang II receptors might not be present in the gerbil brain, with the exception of some binding expressed in the olfactory bulb [5]. The availability of sensitive quantitative autoradiographic techniques made it possible to re-examine this issue. Our interest was stimulated because the gerbil is considered a good experi- mental animal model of stroke and cerebrovascular hy- poxia [7], and Ang II has been reported to play a role in cerebrovascular autoregulation, at least in the rat [14].

In the gerbil brain, the distribution of Ang II binding is more restricted than in the rat and binding could be revealed, in our study, only in a few brain areas. Such a restricted localization can explain why, when membrane preparations from large brain areas were examined, the presence of Ang II receptors was not apparent [5]. Binding is most likely to represent that of the authentic ligand and not of one of its fragments, since no ligand metabolism has been detected by HPLC analysis.

In the gerbil brain, most areas containing substantial amounts of Ang II receptors correspond to structures which, in the rat, contain large numbers of Ang II receptors, and mediate the well known central effects of the peptide (regulation of blood pressure, drinking, salt appetite, and pituitary control). In addition, as it is the case in the rat [20], Ang II receptors are also present in the gerbil inferior olivary complex and in the external plexiform layer of the olfactory bulb, where the Ang II effects are still not fully understood. In the gerbil, nevertheless, the extent of Ang II receptor expression is more restricted than in the rat; for example, no Ang II receptors are present in the gerbil suprachiasmatic nuclei, or in the gerbil major cerebral arteries, while they are very conspicuous in these struc- tures in the rat.

Most of the Ang II receptors in the gerbil brain corre- spond to areas which, in the rat, express only AT 1 receptor subtype [20]. Selective displacement of Ang II binding in the gerbil brain and pituitary gland, however, yielded an unexpected result: none of the Ang II binding could be displaced by the AT 1 selective antagonist losartan. Further- more, in most gerbil areas, the AT 2 selective ligand CGP 42112 was also unable to displace Ang II binding. The conclusion from these experiments was that gerbil brain and pituitary receptors were not of the AT 1 or the AT 2 subtypes, at least from the point of view of their affinity profiles for selective ligands. The exception was the olfac-

186 A.M. de Olit eira et al. / Brain Research 705 (1995) 177-187

tory bulb, where Ang II binding, displaced by CGP 42112, could be considered to belong to the AT 2 subtype.

The expression of relatively large numbers of Ang II receptors in the gerbil hippocampus was of particular interest, because of the sensitivity of hippocampal neurons to hypoxia [16], the proposed role of Ang II in the regulation of cerebral circulation [14] and in hippocampal function and memory [23], and the value of the gerbil model for the study of cerebrovascular function [4]. For these reasons we focused further receptor characterization in this area.

We describe the presence of a large number of sat- urable, high affinity binding sites to [125I]Sarl-Ang II in the gerbil hippocampus. The binding sites are selectively localized to specific hippocampal structures, with an an- tero-posterior, medial-lateral gradient. Displacement by the agonists Ang II and Ang III but not by the antagonist Sar~-IleS-Ang II shows the presence of two binding sites. These sites may correspond to different affinity states of the same receptor, but this is not presently clarified.

Ang II binding to gerbil hippocampus is inhibited by more than 90% by unlabeled Ang II, but it is not sensitive to incubation in the presence of the AT~ receptor antago- nist losartan, or the AT 2 receptor ligand CGP 42112. Our results indicate that the gerbil hippocampal binding sites are Ang II sites, but pharmacologically distinct from the classical Ang II receptor subtypes AT 1 and AT2 This finding contrasts with [t25I]Sar~-Ang II binding in the rat hippocampus, which is displaced by losartan, and can be characterized as ATj subtype [20].

Several lines of evidence demonstrate that the Ang II receptors described here are distinct from these binding sites recently described for Ang IV (AT 4) in the guinea pig hippocampus [6,11,18,24], and the AT 4 binding sites de- scribed here. First, Ang II receptors are recognized by Ang II, Ang III, and Sar 1, IleS-Ang II with high affinity, and the binding is insensitive to displacement with unlabeled Ang IV or other Ang II-derived peptide fragments. In turn, [~2SI]Ang IV is displaced with high affinity only by unla- beled Ang IV, and binding to this AT 4 site is not affected by incubation in the presence of GTP analogues [11]. Second, the Ang II sites are only expressed on those hippocampal layers and structures where AT 4 binding sites are absent, as determined in consecutive sections run in parallel. In fact, binding to Ang II is localized to the stratum oriens, stratum radiatum and the lacunar molecular layers of the CA1 subfield, and to the molecular layer of the gyms dentatus. Conversely, Ang IV binding is ex- pressed only in the pyramidal layer of the CA1, CA2 and CA3 subfields, and in the granular molecular layer of the gyrus dentatus. Thus, in the gerbil, Ang II and Ang IV binding appear to be precisely intercalated from each other in adjacent hippocampal layers. Third, AT 4 binding sites have been reported to be widely distributed in the adult guinea pig brain, including, besides the hippocampus, cor- tical areas, the caudate putamen, thalamus, cerebellum and

motor nuclei [11], and we now report a similar widespread distribution in the gerbil brain. Conversely, the distribution of Ang II binding in the gerbil brain is very restricted, and, within the hippocampus, is predominantly localized to its anterior part.

In addition, the gerbil hippocampal Ang II binding site is not similar to any of the other recently discovered Ang II receptors. For example, the amphibian Ang II receptor expresses a very high affinity for CGP 42112 [9]; the Ang II receptor from the turkey adrenal gland has a distinct affinity for Ang II peptides, different from the gerbil Ang II receptor [12]; the rat AT3receptor, as proposed by Sandberg et al. [15] and the mesangial cell subtype [25] have a relatively high affinity for losartan. In addition, the gerbil Ang II receptor described here is not similar to that described by Chaki and Inagami [3], since binding to that receptor is not displaced by Ang III nor sensitive to GTP analogues.

According to the standard nomenclature [2], Ang II binding to a given tissue which is not displaced with high affinity by the AT~ antagonists or the AT 2 selective lig- ands should be considered at the present time 'atypical'.

The function of the atypical gerbil hippocampal Ang II site is a matter of speculation. The selective localization to hippocampal structures suggests a possible role in memory mechanisms. The nature of the possible transduction mech- anisms for this binding site is also a matter of speculation. However, the sensitivity of the gerbil hippocampal Ang II binding to guanine nucleotides suggests an association with G-proteins. The clarification of the function and structure of the novel receptor described here will require further physiological and behavioral studies.

The present results add additional complexity to the current characterization and classification of Ang II recep- tors. The revelation of an ever increasing variability and complexity of Ang II receptors, of alternative synthetic pathways for Ang II [14] and of possible new related active peptides, suggests that the angiotensin system is more complex, widespread and variable than proposed earlier, and that the system may participate in many func- tions other than the classical modulation of the central autonomic system.

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