Proc. Nati. Acad. Sci. USA Vol. 81, pp. 1575-1579, March 1984 Neurobiology Autoradiographic localization of angiotensin II receptors in rat brain (peptide neurotransmitter/renin/circumventricular organs/hypothalamus/blood pressure) FREDERICK A. 0. MENDELSOHN*t, REMI QUIRIONt§, JUAN M. SAAVEDRA¶, GRETI AGUILERA*, AND KEVIN J. CATT* *Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development; tExperimental Therapeutics Branch, National Institute of Neurological and Communicative Disorders and Stroke and Section of Brain Biochemistry, Neuroscience Branch, National Institute of Mental Health; and ¶Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20205 Communicated by Irvine H. Page, October 11, 1983 ABSTRACT The 125I-labeled agonist analog [1-sarcosine]- it was found mainly in the thalamus, hypothalamus, mid- angiotensin II ([Sar']AII) bound with high specificity and af- brain, septum, and medulla (23, 25), with highest concentra- finity (Ka = 2 x 109 M-1) to a single class of receptor sites in tions in the lateral septum, superior colliculus, and area pos- rat brain. This ligand was used to analyze the distribution of trema (26, 28). Binding sites for blood-borne All have been All receptors in rat brain by in vitro autoradiography followed localized in the circumventricular organs, particularly the by computerized densitometry and color coding. A very high subfornical organ (29), and, after All injection into the cere- density of All receptors was found in the subfornical organ, bral ventricles, in the organum vasculosum of the lamina ter- paraventricular and periventricular nuclei of the hypothala- minalis (30). In such studies, labeling of the respective sites mus, nucleus of the tractus solitarius, and area postrema. A depends on access of All to the brain after intravenous or high concentration of receptors was found in the suprachias- intracerebroventricular injection as well as the presence of matic nucleus of the hypothalamus, lateral olfactory tracts, specific binding sites. Because the detailed anatomical distri- nuclei of the accessory and lateral olfactory tracts, triangular bution of All receptors in the brain has not been described, septal nucleus, subthalamic nucleus, locus coeruleus, and infe- we have localized All receptors in rat brain by an in vitro rior olivary nuclei. Moderate receptor concentrations were autoradiographic technique (31, 32). found in the organum vasculosum of the lamina terminalis, median preoptic nucleus, medial habenular nucleus, lateral MATERIALS AND METHODS septum, ventroposterior thalamic nucleus, median eminence, medial geniculate nucleus, superior colliculus, subiculum, pre- 125I-labeled [1-sarcosine]AII (125I-[Sar']AII) was prepared by and parasubiculum, and spinal trigeminal tract. Low concen- modified Chloramine-T radioiodination (33) of the potent trations of sites were seen in caudate-putamen, nucleus accum- All agonist [Sar']AII, which was generously provided by M. bens, amygdala, and gray matter of the spinal cord. These Khosla (Cleveland Clinic, Ohio). The product was purified studies have demonstrated that All receptors are distributed by HPLC on a C-18 column in acetonitrile/0.1 M ammonium in a highly characteristic anatomical pattern in the brain. The bicarbonate buffer, pH 8.0 (15:85) and had a specific activity high concentrations of All receptors at numerous physiologi- of 800 p.Ci/lsg (1 Ci = 37 GBq) as determined by self-dis- cally relevant sites are consistent with the emerging evidence placement in a radioligand-receptor assay using rat adrenal for multiple roles of All as a neuropeptide in the central ner- homogenate (34). vous system. Characterization of the brain binding sites was performed on a block of tissue including the hypothalamus, thalamus, septum, and midbrain, because this area is known to contain In addition to its well-defined peripheral actions in smooth a relatively high concentration of All receptors (25). The muscle and the adrenal gland, angiotensin II (All) has impor- brain tissue was homogenized in 10 vol of ice-cold 20 mM tant actions in the central nervous system. These include NaHCO3 and the 1,000-30,000 x g fraction was suspended stimulation of drinking, increased salt appetite, elevation of in 10 mM sodium phosphate, pH 7.4/120 mM NaCl/5 mM blood pressure (1-3), and release of several pituitary hor- Na2EDTA/0.1 mM bacitracin/0.2% bovine serum albumin mones, including vasopressin, corticotropin (3-5), and pro- (buffer A) containing 0.1 mM phenylmethylsulfonyl fluoride lactin (6). All components of the renin-angiotensin system and 100 kallikrein units of aprotinin per ml as protease inhibi- (3), including renin (7, 8), angiotensin-converting enzyme (9- tors. Approximately 800 ,ug of "particulate" protein was in- 13), and the polypeptide precursor angiotensinogen (14), cubated in 0.5 ml of buffer A with 1251-[Sar']AII (120 pM) for have been identified in the brain. Renin has been localized in 1 hr at 20°C. Competing ligands were [Sar']AII, All, des- neurones of the medulla oblongata, cerebellar nuclei, and Asp'-AII, and angiotensin I. Nonspecific binding was deter- hypothalamus (15) and in cells of the anterior pituitary (16). mined in the presence of 1 ,uM All. After incubation, free All-containing nerve terminals and neurones have been lo- and bound tracer was separated by filtration through glass calized in brain by immunohistochemistry (17-22). High den- fiber discs and bound radioactivity was measured in a Beck- sities were found in the substantia gelatinosa of the spinal man y spectrometer. The All binding data were analyzed by cord and spinal trigeminal nucleus, sympathetic lateral col- a nonlinear model-fitting computer program (35). umn and medial external layer of the median eminence (17), For autoradiography, male Sprague-Dawley rats of =300 g and hypothalamus (20, 22). were killed by decapitation and the brains were rapidly re- In addition, receptors for All have been clearly identified frozen in at mounted on in membrane fractions of brain (23-28). In calf brain, All moved, 2-methylbutane -40°C, binding was highest in the cerebellum (23), whereas in the rat Abbreviations: All, angiotensin II; Sar, sarcosine. tPresent address: Department of Medicine, University of Mel- The publication costs of this article were defrayed in part by page charge bourne, Austin Hospital, Heidelberg, Victoria 3084, Australia. payment. This article must therefore be hereby marked "advertisement" §Present address: Douglas Hospital Research Centre, 6875 Blvd La- in accordance with 18 U.S.C. §1734 solely to indicate this fact. Salle, Verdun, PQ H4H IR3, Canada. 1575 Downloaded by guest on September 30, 2021 1576 Neurobiology: Mendelsohn et al. Proc. NaM Acad Sci. USA 81 (1984) chucks, and cut into 25-gam-thick sections in a cryostat at the tractus solitarius (Fig. 2 g and h), and area postrema (Fig. -14'C. The sections were thaw-mounted on gelatin-coated 2h). In the paraventricular nucleus, very high levels of All slides, dried in a desiccator at 40C for 2 hr, and stored at binding sites were seen in both the magnocellular and parvo- -80'C (31). The slide-mounted sections were preincubated cellular regions. in 5 ml of buffer (10 mM sodium phosphate, pH 7.4/120 mM High densities of All receptors were present in the lateral NaCl/5 mM Na2EDTA/0.1 mM bacitracin/0.2% bovine se- olfactory tract (Fig. 2a), nucleus of the lateral olfactory tract rum albumin) for 15 min at 20'C and then incubated with 1251_ (Fig. 2j), nucleus of the accessory olfactory tract (Fig. 2j), [Sar']AII (-160 pM) in 5 ml of fresh buffer for 1 hr at 20'C. olfactory tubercule (Fig. 2a), suprachiasmatic nucleus of the Nonspecific binding was determined in the presence of 1 ,xM hypothalamus (Fig. 2j), triangular septal nucleus (Fig. 2b), All. The slides were then transferred through four 60-sec subthalamic nucleus (Fig. 21), locus coeruleus (Fig. 2f), and successive changes of ice-cold 50 mM Tris'HCl buffer (pH inferior olivary nuclei (Fig. 2 g and h). Moderate densities of 7.4) to remove nonspecifically bound ligand. At the end of binding sites were seen in the organum vasculosum of the the rinsing period, the slides were rapidly dried under a lamina terminalis (not shown), median preoptic nucleus (nucle- stream of cold air and placed in cassettes (Wolf X-Ray, West us medianus), median eminence (not shown), medial habenular Hempstead, NY) for exposure to LKB Ultrofilm for 4 days nucleus (Fig. 2k), superior colliculus (Fig. 2e), ventroposterior at room temperature. The films were then processed and thalamic nucleus (not shown), hippocampus (Fig. 2e), mam- grain density was quantitated by computerized densitometry millary nuclei (Fig. 21), subiculum (not shown), pre- and par- with color-coded image analysis (36). asubiculum (Fig. 2e), lateral septum (not shown), medial ge- niculate nucleus (Fig. 2e), and spinal trigeminal tract (not RESULTS shown). Low concentrations of sites were seen in the cauda- Characteristics of [Sar'jAII Binding Sites. In brain mem- teputamen (Fig. 2 b and j), nucleus accumbens (Fig. 2a), branes prepared from the hypothalamus-thalamus-septum- amygdala (not shown), and gray matter of the spinal cord midbrain area, [Sar']AII bound to a single class of high-affin- (Fig. 2i). Very low receptor levels were found in most corti- ity sites with a Ka of 2.0 + 0.4 x 109 M-1 and concentration cal areas (Fig. 2 a-e, j, and 1), globus pallidus (not shown), of 13 ± 2 fmol/mg of protein. The relative potencies of angi- hippocampus (Fig. 2e), cerebellum (Fig. 2 f-h), and white otensin analogs in displacing 1251-[Sar1]AII were as follows: matter such as corpus callosum (Fig. 2 a-c). [Sar1]AII, 1.00; All, 0.17; des-Aspl-AII, 0.12; angiotensin I, 0.0017 (Fig. 1). DISCUSSION Autoradiographic Studies. Angiotensin receptors were found in several discrete brain regions, which were classified The potent angiotensin radioligand 1251-[Sar']AII bound to a after computerized densitometry into the following groups.