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Cellular localization of angiotensin type 1 receptor and angiotensinogen mRNAs in the subfornical organ of the rat brain
Neuroscience Letters, 150 (1993) 153-158
153
Elsevier Scientific Publishers Ireland Ltd.
NSL 09285
Cellular localization of angiotensin type 1 receptor and angiotensinogen mRNAs in the subfornical organ of the rat brain Andrea Lippoldt% Bernd B u n n e m a n n b, N a o h a r u Iwai% Rainer Metzger d, Tadashi Inagami c, Kjell Fuxe b and Detlev Ganten a °Max-Delbriick-Centerfor Molecular Medicine [MDC], Berlin-Buch (FRG), bDepartment of Histology and Neurobiology, Karolinska Institute, Stockholm (Sweden), ' Vanderbilt University School of Medicine, Nashville, TN (USA) and dDepartment of Pharmacology and German Institute of High Blood Pressure Research, Heidelberg (FRG) (Received 8 August 1992; Revised version received 5 November 1992; Accepted 6 November 1992)
Key words: Brain; Angiotensin type 1 receptor mRNA; Angiotensinogen mRNA; Glial fibrillary acidic protein immunocytochemistry; In situ hybridization; Rat The cellular localization of angiotensin type l receptor (AT 1) and angiotensinogen mRNA expression in the subfornical organ (SFO) of the rat brain has been studied by means of non-radioactive in situ hybridization combined with immunocytochemistry for glial fibrillary acidic protein (GFAP) and Neutral red staining. The AT 1 receptor mRNA expression is shown to be within putative nerve cells without any association with the glial fibrillary acidic protein (GFAP)-immunoreactive (IR) cells. In contrast the angiotensinogen cRNA expression is associated predominantly with GFAP-IR cells. The results demonstrate that a neuronal AT 1 receptor mediates the actions of circulating angiotensin II on the SFO and that the angiotensinogen mRNA is predominantly expressed in the SFO astroglial cells.
The subfornical organ (SFO) plays a crucial role in blood pressure regulation and body fluid homeostasis. It is located outside the blood-brain barrier and exposed to changes in the peripheral renin angiotensin system (RAS). Using biochemical binding studies [16] and receptor autoradiography with selective AT 1 and AT 2 receptor antagonists [9, 20], the SFO was shown to contain the highest number of specific All binding sites, but to involve exclusively the AT 1 receptor subtype [8, 9, 25]. Evidence exists for an increased number of AII binding sites in the SFO of brains of hypertensive rats (SHR) [23, 24], and that the angiotensin-converting enzyme (ACE) inhibitor enalapril given orally can produce a 50% decrease in All receptors in the SFO of SHR [22]. These results emphasize an influence of the peripheral RAS on the brain RAS [7] via the SFO in maintaining and regulating blood pressure. Recently, high levels of the AT 1 receptor mRNA have been demonstrated in SFO in the adult male rat brain by autoradiographic in situ hybridization (ISH) [4]. In the present study, the neuronal localization of the SFO AT 1 Correspondence: K. Fuxe, Department of Histology and Neurobiology, Karolinska Institute, Box 60 400, S-10401 Stockholm, Sweden. Fax: (46) 8-33 79 41.
receptor mRNA is demonstrated by non-radioactive in situ hybridization in combination with GFAP immunohistochemistry, while with the same techniques the SFO angiotensinogen mRNA is found to be predominantly expressed in the astroglia. Ten specific pathogen-free adult male SpragueDawley rats (200 g b.wt.) were used in the experiments. The animals were kept under regular lighting conditions (lights on 06.00 h and off20.00 h) and a constant temperature (23°C) with free access to food pellets and tap water. For ISH the animals were anesthetized with sodium pentobarbital (60 mg/kg b.wt.) and perfused through the left heart ventricle with 200 ml of ice-cold 0.9% saline. The brains were snap-frozen with CO2 and 12-/1m-thick coronal sections were made in a cryostat and thawmounted onto poly-L-lysine-coated slides. The RNA probes were synthesized by in vitro transcription from a 733 base pairs (bp) EcoRIIKpnI rat AT 1 receptor subtype-cDNA fragment, representing the positions 86-819 and subcloned into the vector pGEM 3 (Promega, Madison, WI, USA). For linearization of the plasmid StyI was used. Restriction with StyI resulted in two different fragments. These fragments were used for transcription with T 7 or SP 6 RNA polymerase, respec-
154 tively. The transcription with T 7 R N A polymerase (Boehringer, Mannheim, F R G ) resulted in a 353 nucleotide long cRNA, whereas a 380 nucleotide long m R N A was obtained after in vitro transcription using SP 6 R N A polymerase (Boehringer, Mannheim, FRG). The labelling was performed with digoxygenin-11-UTP ( D I G l l-UTP) (Boehringer, Mannheim, FRG). The transcripts were ethanol-precipitated and the R N A concentration was measured in a UV-spectrophotometer at 260 rim. The labelling quality was checked by separating the probes via a denaturating formaldehyde gel electrophoresis, transferring the R N A onto a nylon membrane (Nytran N, Schleicher and Schiill, F R G ) and detection of the digoxygenin by an alkaline phosphatase-conjugated antibody (Boehringer, Mannheim, FRG). The specificity of the probes was shown by RNase protection assay [4]. The slides were brought to r o o m temperature (RT) and fixed for 5 min in 3% paraformaldehyde in phosphate-buffered saline (PBS) pH 7.0. After fixation the slides were washed in PBS for 10 min and rinsed two times in sterilized water for 5 min, each followed by a deproteination of the tissue with 0.2 M HCI for 10 min. The slides were rinsed again 2 × 3 min each in PBS. To decrease background, the tissue was acetylated in 0.1 M triethanolamine p H 8.0/0.25% acetic anhydride for 20 min, washed again briefly in PBS, dehydrated in graded ethanol and air-dried. The slides were prehybridized in a humidified chamber with 150/~1 prehybridization buffer (50% deionized formamide, 50 m M Tris-HC1 p H 7.6, 25 mM E D T A p H 8.0, 20 m M NaCI, 0.25 mg/ml yeast tRNA, 2.5 x Denhardt's solution (0.05% Ficoll, 0.05% polyvinylpyrrolidone, 0.05% bovine serum albumin)) for 24h. After draining the prehybridization buffer off the slides, the sections were hybridized with 15 ¢tl hybridization buffer (50% deionized formamide, 20 m M Tris-HCl pH 7.6, 1 m M E D T A pH 8.0, 0.3 M NaC1, 0.2 M DTT, 0.5 mg/ml yeast t R N A , 0.1 mg/ml poly-A-RNA, 1× Denhardt's solution, 10% dextransulfate) containing either the labelled c R N A or m R N A for control experiments. The sections were covered with siliconized coverslips and incubated at 37°C for 18 h in a humidified
chamber. The coverslips were removed by washing with 1 x standard saline citrate (SSC) at 48°C for 30 min, followed by washing in 0.5 × SSC/50% formamide at 48°C for 4 h and changing the washing solution every hour. The slides were rinsed for 5 min in 1 x SSC and subsequently 2 x 5 min in buffer ! (100 m M Tris-HCl, 150 m M NaCI, pH 7.5 and then incubated for 30 min with 5% normal sheep serum (NSS) in buffer I (150yl tbr each section). The slides were rinsed briefly in buffer 1 and then incubated with 150/A per section sheep antiDIG-alkaline phosphatase antibody (1:300 in buffer I, containing 1% NSS and 0.3% Triton X-100) overnight at 4°C in a humidified chamber. The slides were then rinsed briefly in buffer I and washed 3 × 10 min in buffer l, rinsed in buffer II (100 mM Tris-HC1, 100 m M NaC1, 50 m M MgCI> pH 9.5) and equilibrated with buffer lI tbr 5 min. The color reaction for alkaline phosphatase was performed by incubating the sections in a humidified chamber with 150 ~tl chromogen solution (45/~1 of Nitro blue tetrazolium (75 mg/ml in 70% dimethylformamide) and 35/11 of 5-bromo-4-chloro-3-indolyl-phosphate (50 mg/ml in 100% dimethylformamide)in 10 ml buffer II) at room temperature in the dark for 20 h. The reaction was stopped by immersing the slides in 10 m M Tris-HCl, 1 mM E D T A p H 8.0 for 5 min. The slides were rinsed in distilled water, air-dried, dipped briefly in xylene and coverslipped with Entellan (Merck, Darmstadt, FRG). The specificity of the signal was demonstrated by hybridization with labelled m R N A probes of the same concentration. GFAP-Immunocvtochemistry in combination with 1SH. Immunocytochemistry was performed using the biotin avidin immunoperoxidase procedure and the Vectastain Kit (Vector, Burlingame, USA) and 3-3'-diaminobenzidine tetrahydrochloride (DAB) as chromogen. After stopping the reaction of the alkaline phosphatase the slides were washed 3 × 10 min in PBS. The GFAP-antibody (Boehringer, Mannheim, F R G ) was applied to the sections (100 ~tl/section, 1:30 in PBS/0.3% Triton X-100) and incubated over night at 4°C. After washing 3 × l0 min in PBS the slides were incubated for 1 h at RT with 100 /A/section of the biotinylated secondary antibody
Fig. 1. Non-radioactive in situ hybridization. The coronal cryostate sections have been hybridized with DIG-11-UTP-labelledAT 1 receptor cRNA (a,c,e,f) or with angiotensinogen cRNA (d), respectively, followed by immunocytochemistryfor GFAP (a,d,f). a: specificallyhybridized material (dark blue) (arrowhead) in the form of large granules is found in putative weakly bluish nerve cell bodies surrounding the nuclei without any association with the GFAP-immunoreactivecells and processes (brownish) (thin arrow), b: control hybridization with DIG-I 1-UTP-labelled AT 1 receptor mRNA. No hybridized material is found, c: the specificallyhybridized material exists (dark blue) (arrowhead) in the form of large granules close to large putative neuronal nuclei stained in red by Neutral red, while the surrounding small putative glial nuclei lack an association with the hybridized material, d: the specificsignal for angiotensinogen cRNA (dark blue material, thick arrow) was found in astroglial cells (brownish). The thin arrow shows the GFAP-IR glial processes, e: the arrowhead shows the specifically hybridized material surrounding the same large putative neuronal nucleus shown in (c) but in higher magnification, f: large granules of specificallyhybridized material are found surrounding putative nerve cell nuclei. The arrowhead shows the same nerve cell as in (a), but in higher magnification. Bar = 20/.tm for a ~:: 40 ¢tm for d f.
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(anti-mouse-IgG, Vector, Burlingame, USA, 1:200 in PBS/0.3% Triton X-100). The slides were rinsed again 3 × 10 min in PBS and incubated for 45 min at RT with 100/d/section Vectastain solution (1:100 in PBS). After washing 3 x 10 min in 50 mM Tris-HCl pH 7.4 the staining with DAB (0.2 mg/ml, 0.005% H202 in 50 mM TrisHCI pH 7.4) was performed for 4 min. The reaction was stopped in Tris-HCl pH 8.0, the slides were air-dried, dipped briefly in xylene and coverslipped with Entellan (Merck, Darmstadt, FRG). Neutral red staining for nuclei. The sections were counterstained after in situ hybridization with Neutral red solution (Sigma, St. Louis, USA), air-dried, dipped briefly in xylene and coverslipped with Entellan. A strong hybridization signal was demonstrated with the AT 1 cRNA probe in the SFO. The hybridized material had the appearance of large granules within the cytoplasm of rounded cells, probably representing nerve cells in view of their size, shape and distribution (Fig. la). The control hybridizations with the DIG-11-UTP-labelled AT 1 mRNA probe showed no labelling above the background (Fig. lb). The neuronal and/or glial localization of the hybridized material was also investigated by combining non-radioactive ISH with immunocytochemistry for GFAP. The specifically hybridized dark blue material was found not to be related to the GFAP-IR cells and their processes shown to exist in the SFO (Fig. la,f). The transcription of the AT 1 gene in neuronal cells was further indicated by staining the nuclei with Neutral red after ISH. In these sections, the hybridized material seen with the non-radioactively labelled AT 1 cRNA probe was associated with the large nerve cell nuclei but not with the small glial nuclei (Fig. lc,e). It was found that the specific AT 1 hybridization signal appeared as small to large granules in GFAP-IR-negative putative nerve cells with a perinuclear localization (Fig. If). The strong hybridization signal demonstrated with the angiotensinogen cRNA but not the corresponding mRNA probe in the SFO was located in the astroglial cells, since the hybridized material (dark blue) was present in the cytoplasm of the GFAP-immunoreactive astrocytes (brownish) (Fig. ld). By combining non-radioactive in situ hybridization and GFAP immunocytochemistry or Neutral red staining for nuclei we were able to obtain indications for a neuronal localization of the AT 1 mRNA and to demonstrate angiotensinogen mRNA in astroglial cells in the SFO of the adult rat brain. The specificity of the hybridized material in the SFO was demonstrated by its absence after incubation with the corresponding labelled mRNA probe for the AT 1 receptor as well as for the angiotensinogen. The demonstration of AT 1 mRNA in the SFO using the non-radioactive in situ hybridization
was in good agreement with our earlier results using radioactive in situ hybridization [4]. From angiotensin II receptor binding studies involving receptor subtype characterization, it was also demonstrated that the very strong binding signal detected in the SFO is linked to the AT 1 receptor [9, 25]. Also in the other areas of the brain related to dipsogenic, cardiovascular and endocrine functions, the AT 1 receptor subtype predominates [9, 25, 31]. However, in neuronal cultures the predominating angiotensin II receptor is of the AT 2 type while in cultured astrocytes the AT 1 receptor appears to dominate [27, 28]. These findings are in contrast to our demonstration of AT 1 receptor mRNA in putative neuronal cells in the SFO of the rat brain. One explanation may be the use of primary cultures derived from 1-dayold or 2-week-old rats [26, 28], since a development-dependent change in the expression of the two receptor subtypes may take place [31]. These findings as well as the demonstration of increased expression of AT 2 receptor in the brain of young rats [31] and the demonstration of only low amounts of AT 1 sites in cultured neonatal neurons [28] might suggest a growth and developmentalrelated regulation of the angiotensin II receptor subtype expression in neuronal cells. The demonstration of a predominant neuronal localization of the AT 1 receptor mRNA in the SFO with angiotensinogen mRNA mainly found in the astrocytes of the SFO, supports the proposed major role of volume transmission in the central renin angiotensin system [1, 2]. In previous papers we and others have shown that the precursor peptide of All, angiotensinogen, is transcribed predominantly in the astroglial cells [3, 5, 6], whereas the angiotensin II itself is mainly found in neuronal cells and nerve fibers [3, 6, 17]. The SFO is an organ located outside of the bloodbrain barrier and represents one site of action of circulating All. It may be stimulated by blood born All but also gives rise to AII-immunoreactive projections to the median preoptic nucleus and the paraventricular nucleus of the hypothalamus and receives an All-positive neuronal input from the lateral hypothalamic area [17, 18]. Moreover predominately astroglial cells of the SFO produce angiotensinogen. The question arises of the role of the SFO in linking the peripheral and the central renin angiotensin systems to one another. A major implication of the present paper is that mainly the AT 1 receptor producing SFO nerve cells may be direct targets for circulating All. Thus, All can bind to the neuronal AT 1 receptor of the SFO and induce excitatory actions on the All-positive neuronal output to the hypothalamic neurosecretory neurons [13]. The All in this subfornical output system may act as neurotransmitter in the preoptic region and the paraventricular hypothalamic nucleus
157 ( P V N ) [17-19] to c o n t r o l b l o o d p r e s s u r e a n d b o d y fluid h o m e o s t a s i s b y releasing c o r t i c o t r o p i n - r e l e a s i n g f a c t o r a n d v a s o p r e s s i n a n d to induce d r i n k i n g b e h a v i o r [10, 11, 21, 29]. W i t h r e g a r d to n e u r o n a l f e e d b a c k s to the S F O T a n a k a et al. [30] suggested t h a t the A l l p r o j e c t i o n s to the S F O f r o m the l a t e r a l h y p o t h a l a m i c a r e a c o u l d h a v e an e x c i t a t o r y influence o n the activity o f n e u r o n s in regions o f the S F O with efferent p r o j e c t i o n s to the P V N [18, 30]. T h e l a t e r a l h y p o t h a l a m i c a r e a p l a y s a role in ingestive b e h a v i o r i n c l u d i n g a n g i o t e n s i n - i n d u c e d thirst [6]. T h e s e A I I i n p u t n e u r o n s m a y t h e r e f o r e t r a n s f e r relev a n t i n f o r m a t i o n to the A T 1-positive S F O neurons. It m a y d o so b y c o n t r o l l i n g the b i n d i n g c h a r a c t e r i s t i c s a n d t r a n s d u c t i o n o f the n e u r o n a l A T 1 r e c e p t o r in the S F O . T h e high c o n c e n t r a t i o n s o f n e u r o n a l l y released A l l m a y desensitize the A T 1 r e c e p t o r o f the S F O n e u r o n s , so t h a t c i r c u l a t i n g A l l p r e s e n t in lower c o n c e n t r a t i o n s n o l o n g e r c a n effectively activate the high affinity A T 1 r e c e p t o r o f the S F O . This results in a r e d u c t i o n in the a b i l i t y o f circ u l a t i n g A l l to increase v a s o p r e s s i n a n d c o r t i c o t r o p i n releasing f a c t o r release [15, 21], a r t e r i a l b l o o d p r e s s u r e [12], a n d to induce thirst [14], m a k i n g p o s s i b l e a negative f e e d b a c k r e g u l a t i o n o f the b l o o d - b o r n A l l signal b y the n e u r o n a l A l l like signal. This s t u d y was s u p p o r t e d b y g r a n t s o f the Swedish M e d i c a l R e s e a r c h C o u n c i l (04X715), the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t ( S F B 317) a n d N I H G r a n t 1 RO1 H L 35821-01. 1 Agnati, L.F., Fuxe, K., Zoli, M., Zini, J., Toffano, G. and Ferraguti, F., A correlation analysis of the regional distribution of central enkephalin and fl-endorphin immunoreactive terminals and of opiate receptors in adult and old male rats. Evidence for the existence of two main types of communication in the central nervous system: the volume transmission and the wiring transmission, Acta Physiol. Scand., 128 (1986) 201-207. 2 Bunnemann, B., Fuxe, K., Bjelke, B. and Ganten, D., The brain renin-angiotensin system and its possible involvement in volume transmission. In K. Fuxe and L.F. Agnati (Eds.), Advances in Neuroscience, Vol. 1, Raven, New York, 1990, pp. 131-158. 3 Bunnemann, B., Fuxe, K., Metzger, R., Bjelke, B. and Ganten, D., The semiquantitative distribution and cellular localization of angiotensinogen mRNA in the rat brain, J. Chem. Neuroanatom., 5 (1992) 245-262. 4 Bunnemann, B., Iwai, N., Metzger, R., Fuxe, K., Inagami, T. and Ganten, D., The distribution of angiotensin II AT 1 receptor subtype mRNA in the rat brain, Neurosci. Lett., 140 (1992) 155-158. 5 Deschepper, C.F., Bouhnik, J. and Ganong, W.F., Localization of angiotensinogen and glial fibrillary acidic protein in astrocytes in rat brain, Brain Res., 374 (1986) 195-198. 6 Fuxe, K., Bunnemann, B., Aronsson, M., Tinner, B., Cintra, A., von Euler, G., Agnati, L.F., Nakanishi, S., Ohkubo, H. and Ganten, D., Pre- and postsynaptic features of the central angiotensin systems. Indications for a role of angiotensin peptides in volume transmission and for interactions with central monoamine neurons, Clin. Exp. Hyp. Theory Pract. A, 10 (Suppl. 1) (1988) 143-168.
7 Fuxe, K., Ganten, D., Bolme, P., Agnati, L.F., H6kfelt, T., Andersson, K., Goldstein, M., Hiiffstrand, A., Unger, T. and Rascher, W., The role of central catecholamine pathways in spontaneous and renal hypertension in rats. In K. Fuxe, M. Goldstein, B. Hrkfelt and T. Hrkfelt (Eds.), Central Adrenaline Neurons, Basic Aspects and Their Role in Cardiovascular Functions, Pergamon, Oxford, 1980, pp. 259-276. 8 Gehlert, D.R., Gackenheimer, S.L. and Schober, D.A., Angiotensin II receptor subtypes in rat brain: dithiothreitol inhibits ligand binding to A II-1 and enhances binding to A II-2, Brain Res., 546 (1991) 161-165. 9 Gehlert, D.R., Gackenheimer, S.L. and Schober, D.A., Autoradiographic localization of subtypes of angiotensin II antagonist binding in the rat brain, Neuroscience, 44 (1991) 501-514. 10 Iovino, M. and Steardo, L., Vasopressin release to central and peripheral angiotensin II in rats with lesions of the subfornical organ, Brain Res., 322 (1984) 365-368. 11 Iovino, M. and Steardo, L., Thirst and vasopressin secretion following central administration of angiotensin II in rats with lesions of the septal area and subfornical organ, Neuroscience, 15 (1985) 6165. 12 Ishibashi, S. and Nicolaides, S., Hypertension induced by electrical stimulation of the subfornical organ (SFO), Brain Res. Bull., 6 (1980) 135-139. 13 Jhamadas, J.H., Lind, R.W. and Renaud, L.P., Angiotensin II may mediate excitatory neurotransmission from the subfornical organ to the hypothalamic supraoptic nucleus: an anatomical and electrophysiological study in the rat, Brain Res., 487 (1989) 52-61. 14 Kadekaro, M., Cohen, S., Terrell, M.L., Lekan, H., Gary, H. and Eisenberg, H.M., Independent activation of subfornical organ and hypothalamo-neurohypophysial system during administration of angiotensin II, Peptides, 10 (1989) 423-429. 15 Knepel, W., Nutto, D. and Meyer, D.K., Effect of subfornical organ efferent projections on vasopressin release induced by angiotensin or isoprenaline in the rat, Brain Res., 248 (1982) 180-184. 16 Leung, K.W., Smith, R.D., Timmermanns, P.B.M.W.M. and Chin, A.T., Regional distribution of the two subtypes of angiotensin II receptor in rat brain using selective nonpeptide antagonists, Neurosci. Lett., 123 (1991) 95-98. 17 Lind, R.W., Swanson, L.W. and Ganten, D., Organization of angiotensin II immunoreactive cells and fibers in the rat central nervous system, Neuroendocrinology, 40 (1985) 2-24. 18 Lind, R.W. and Ganten, D., Angiotensin. In A. Bj6rklund, T. H6kfelt and M.J. Kuhar (Eds.), Neuropeptides in the CNS, Part II, Handbook of Chemical Neuroanatomy, Vol. 9, Elsevier, Amsterdam, 1990, pp. 165-286. 19 Lind, R.W., Swanson, L.W. and Ganten, D., Angiotensin II immunoreactivity in the neural afferents and efferents of the subfornical organ of the rat, Brain Res., 321 (1984) 209-215. 20 Mendelsohn, F.A.O., Quision, R., Saavedra, J.M., Aguilera, G. and Catt, K.J., Autoradiographic localization of angiotensin II receptors in the rat brain, Proc. Natl. Acad. Sci. USA, 81 (1984) 15751579. 21 Murakami, K. and Ganong, W.F., Site at which angiotensin II acts to stimulate ACTH secretion in vivo, Neuroendocrinology, 46 (1987) 231-235. 22 Nazarali, A.J., Gutkind, J.S., Correa, F.M.A. and Saavedra, J.M., Decreased angiotensin II receptors in the subfornical organ of spontaneously hypertensive rats after chronic antihypertensive treatment with enalapril, AJH, 3 (1990) 59~51. 23 Saavedra, J.M., Correa, F.M.A., Plunkett, L.M., Israel, A., Kurikara, M. and Shigematsu, K., Binding of angiotensin and atrial
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nutriuretic peptide in brain of hypertensive rats, Nature, 320 (1986) 758-760. Saavedra, J.M., Correa, F.M.A., Kurihara, M. and Shigematsu, K., Increased number of angiotensin II receptors in the subfornical organ of spontaneously hypertensive rats, J. Hypertens., 4 (Suppl. 5) (1986) $27-$30. Speth, R.C., Rowe, B.P., Grove, K.L., Carter, M.R. and Saylor, D., Sulfhydryl reducing agents distinguish two subtypes of angiotensin II receptors in the rat brain, Brain Res., 548 (1991) 1-8. Sumners, C., Myers, L.M., Kalberg, C.J. and Raizada, M.K., Physiological and pharmacological comparisons of angiotensin II receptors in neuronal astrocyte glial cultures, Prog. Neurobiol., 34 (1990) 355-385. Sumners, C. and Myers, L.M., Angiotensin II decreases cGMP levels in neuronal cultures from rat brain, Am. J. Physiol., 260 (1991) C79-C87.
28 Sumners, C., Tang, W., Zelezna, B. and Raizada, M.K., Angiotensin II receptor subtypes are coupled with distinct signal-transduction mechanisms in neurons and astrocytes from rat brain, Proc. Natl. Acad. Sci. USA, 88 (1991) 7567-7571. 29 Swanson, L.W., Sawchenko, P.E., Rivier, J. and Vale, W.W., Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study, Neuroendocrinology, 36 (1983) 165 186. 30 Tanaka, J., Kaba, H., Saito, H. and Seto, K., Lateral hypothalamic area stimulation excites neurons in the region of the subfornical organ with efferent projections to the hypothalamic paraventricular nucleus in the rat, Brain Res., 379 (1986) 200 203. 31 Tsutsumi, K. and Saavedra, J.M., Characterization and development of angiotensin II receptor subtypes (AT I and AT 2) in rat brain, Am. J. Physiol., 261 (1991) R209-R216.
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Elsevier Scientific Publishers Ireland Ltd.
NSL 09285
Cellular localization of angiotensin type 1 receptor and angiotensinogen mRNAs in the subfornical organ of the rat brain Andrea Lippoldt% Bernd B u n n e m a n n b, N a o h a r u Iwai% Rainer Metzger d, Tadashi Inagami c, Kjell Fuxe b and Detlev Ganten a °Max-Delbriick-Centerfor Molecular Medicine [MDC], Berlin-Buch (FRG), bDepartment of Histology and Neurobiology, Karolinska Institute, Stockholm (Sweden), ' Vanderbilt University School of Medicine, Nashville, TN (USA) and dDepartment of Pharmacology and German Institute of High Blood Pressure Research, Heidelberg (FRG) (Received 8 August 1992; Revised version received 5 November 1992; Accepted 6 November 1992)
Key words: Brain; Angiotensin type 1 receptor mRNA; Angiotensinogen mRNA; Glial fibrillary acidic protein immunocytochemistry; In situ hybridization; Rat The cellular localization of angiotensin type l receptor (AT 1) and angiotensinogen mRNA expression in the subfornical organ (SFO) of the rat brain has been studied by means of non-radioactive in situ hybridization combined with immunocytochemistry for glial fibrillary acidic protein (GFAP) and Neutral red staining. The AT 1 receptor mRNA expression is shown to be within putative nerve cells without any association with the glial fibrillary acidic protein (GFAP)-immunoreactive (IR) cells. In contrast the angiotensinogen cRNA expression is associated predominantly with GFAP-IR cells. The results demonstrate that a neuronal AT 1 receptor mediates the actions of circulating angiotensin II on the SFO and that the angiotensinogen mRNA is predominantly expressed in the SFO astroglial cells.
The subfornical organ (SFO) plays a crucial role in blood pressure regulation and body fluid homeostasis. It is located outside the blood-brain barrier and exposed to changes in the peripheral renin angiotensin system (RAS). Using biochemical binding studies [16] and receptor autoradiography with selective AT 1 and AT 2 receptor antagonists [9, 20], the SFO was shown to contain the highest number of specific All binding sites, but to involve exclusively the AT 1 receptor subtype [8, 9, 25]. Evidence exists for an increased number of AII binding sites in the SFO of brains of hypertensive rats (SHR) [23, 24], and that the angiotensin-converting enzyme (ACE) inhibitor enalapril given orally can produce a 50% decrease in All receptors in the SFO of SHR [22]. These results emphasize an influence of the peripheral RAS on the brain RAS [7] via the SFO in maintaining and regulating blood pressure. Recently, high levels of the AT 1 receptor mRNA have been demonstrated in SFO in the adult male rat brain by autoradiographic in situ hybridization (ISH) [4]. In the present study, the neuronal localization of the SFO AT 1 Correspondence: K. Fuxe, Department of Histology and Neurobiology, Karolinska Institute, Box 60 400, S-10401 Stockholm, Sweden. Fax: (46) 8-33 79 41.
receptor mRNA is demonstrated by non-radioactive in situ hybridization in combination with GFAP immunohistochemistry, while with the same techniques the SFO angiotensinogen mRNA is found to be predominantly expressed in the astroglia. Ten specific pathogen-free adult male SpragueDawley rats (200 g b.wt.) were used in the experiments. The animals were kept under regular lighting conditions (lights on 06.00 h and off20.00 h) and a constant temperature (23°C) with free access to food pellets and tap water. For ISH the animals were anesthetized with sodium pentobarbital (60 mg/kg b.wt.) and perfused through the left heart ventricle with 200 ml of ice-cold 0.9% saline. The brains were snap-frozen with CO2 and 12-/1m-thick coronal sections were made in a cryostat and thawmounted onto poly-L-lysine-coated slides. The RNA probes were synthesized by in vitro transcription from a 733 base pairs (bp) EcoRIIKpnI rat AT 1 receptor subtype-cDNA fragment, representing the positions 86-819 and subcloned into the vector pGEM 3 (Promega, Madison, WI, USA). For linearization of the plasmid StyI was used. Restriction with StyI resulted in two different fragments. These fragments were used for transcription with T 7 or SP 6 RNA polymerase, respec-
154 tively. The transcription with T 7 R N A polymerase (Boehringer, Mannheim, F R G ) resulted in a 353 nucleotide long cRNA, whereas a 380 nucleotide long m R N A was obtained after in vitro transcription using SP 6 R N A polymerase (Boehringer, Mannheim, FRG). The labelling was performed with digoxygenin-11-UTP ( D I G l l-UTP) (Boehringer, Mannheim, FRG). The transcripts were ethanol-precipitated and the R N A concentration was measured in a UV-spectrophotometer at 260 rim. The labelling quality was checked by separating the probes via a denaturating formaldehyde gel electrophoresis, transferring the R N A onto a nylon membrane (Nytran N, Schleicher and Schiill, F R G ) and detection of the digoxygenin by an alkaline phosphatase-conjugated antibody (Boehringer, Mannheim, FRG). The specificity of the probes was shown by RNase protection assay [4]. The slides were brought to r o o m temperature (RT) and fixed for 5 min in 3% paraformaldehyde in phosphate-buffered saline (PBS) pH 7.0. After fixation the slides were washed in PBS for 10 min and rinsed two times in sterilized water for 5 min, each followed by a deproteination of the tissue with 0.2 M HCI for 10 min. The slides were rinsed again 2 × 3 min each in PBS. To decrease background, the tissue was acetylated in 0.1 M triethanolamine p H 8.0/0.25% acetic anhydride for 20 min, washed again briefly in PBS, dehydrated in graded ethanol and air-dried. The slides were prehybridized in a humidified chamber with 150/~1 prehybridization buffer (50% deionized formamide, 50 m M Tris-HC1 p H 7.6, 25 mM E D T A p H 8.0, 20 m M NaCI, 0.25 mg/ml yeast tRNA, 2.5 x Denhardt's solution (0.05% Ficoll, 0.05% polyvinylpyrrolidone, 0.05% bovine serum albumin)) for 24h. After draining the prehybridization buffer off the slides, the sections were hybridized with 15 ¢tl hybridization buffer (50% deionized formamide, 20 m M Tris-HCl pH 7.6, 1 m M E D T A pH 8.0, 0.3 M NaC1, 0.2 M DTT, 0.5 mg/ml yeast t R N A , 0.1 mg/ml poly-A-RNA, 1× Denhardt's solution, 10% dextransulfate) containing either the labelled c R N A or m R N A for control experiments. The sections were covered with siliconized coverslips and incubated at 37°C for 18 h in a humidified
chamber. The coverslips were removed by washing with 1 x standard saline citrate (SSC) at 48°C for 30 min, followed by washing in 0.5 × SSC/50% formamide at 48°C for 4 h and changing the washing solution every hour. The slides were rinsed for 5 min in 1 x SSC and subsequently 2 x 5 min in buffer ! (100 m M Tris-HCl, 150 m M NaCI, pH 7.5 and then incubated for 30 min with 5% normal sheep serum (NSS) in buffer I (150yl tbr each section). The slides were rinsed briefly in buffer 1 and then incubated with 150/A per section sheep antiDIG-alkaline phosphatase antibody (1:300 in buffer I, containing 1% NSS and 0.3% Triton X-100) overnight at 4°C in a humidified chamber. The slides were then rinsed briefly in buffer I and washed 3 × 10 min in buffer l, rinsed in buffer II (100 mM Tris-HC1, 100 m M NaC1, 50 m M MgCI> pH 9.5) and equilibrated with buffer lI tbr 5 min. The color reaction for alkaline phosphatase was performed by incubating the sections in a humidified chamber with 150 ~tl chromogen solution (45/~1 of Nitro blue tetrazolium (75 mg/ml in 70% dimethylformamide) and 35/11 of 5-bromo-4-chloro-3-indolyl-phosphate (50 mg/ml in 100% dimethylformamide)in 10 ml buffer II) at room temperature in the dark for 20 h. The reaction was stopped by immersing the slides in 10 m M Tris-HCl, 1 mM E D T A p H 8.0 for 5 min. The slides were rinsed in distilled water, air-dried, dipped briefly in xylene and coverslipped with Entellan (Merck, Darmstadt, FRG). The specificity of the signal was demonstrated by hybridization with labelled m R N A probes of the same concentration. GFAP-Immunocvtochemistry in combination with 1SH. Immunocytochemistry was performed using the biotin avidin immunoperoxidase procedure and the Vectastain Kit (Vector, Burlingame, USA) and 3-3'-diaminobenzidine tetrahydrochloride (DAB) as chromogen. After stopping the reaction of the alkaline phosphatase the slides were washed 3 × 10 min in PBS. The GFAP-antibody (Boehringer, Mannheim, F R G ) was applied to the sections (100 ~tl/section, 1:30 in PBS/0.3% Triton X-100) and incubated over night at 4°C. After washing 3 × l0 min in PBS the slides were incubated for 1 h at RT with 100 /A/section of the biotinylated secondary antibody
Fig. 1. Non-radioactive in situ hybridization. The coronal cryostate sections have been hybridized with DIG-11-UTP-labelledAT 1 receptor cRNA (a,c,e,f) or with angiotensinogen cRNA (d), respectively, followed by immunocytochemistryfor GFAP (a,d,f). a: specificallyhybridized material (dark blue) (arrowhead) in the form of large granules is found in putative weakly bluish nerve cell bodies surrounding the nuclei without any association with the GFAP-immunoreactivecells and processes (brownish) (thin arrow), b: control hybridization with DIG-I 1-UTP-labelled AT 1 receptor mRNA. No hybridized material is found, c: the specificallyhybridized material exists (dark blue) (arrowhead) in the form of large granules close to large putative neuronal nuclei stained in red by Neutral red, while the surrounding small putative glial nuclei lack an association with the hybridized material, d: the specificsignal for angiotensinogen cRNA (dark blue material, thick arrow) was found in astroglial cells (brownish). The thin arrow shows the GFAP-IR glial processes, e: the arrowhead shows the specifically hybridized material surrounding the same large putative neuronal nucleus shown in (c) but in higher magnification, f: large granules of specificallyhybridized material are found surrounding putative nerve cell nuclei. The arrowhead shows the same nerve cell as in (a), but in higher magnification. Bar = 20/.tm for a ~:: 40 ¢tm for d f.
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(anti-mouse-IgG, Vector, Burlingame, USA, 1:200 in PBS/0.3% Triton X-100). The slides were rinsed again 3 × 10 min in PBS and incubated for 45 min at RT with 100/d/section Vectastain solution (1:100 in PBS). After washing 3 x 10 min in 50 mM Tris-HCl pH 7.4 the staining with DAB (0.2 mg/ml, 0.005% H202 in 50 mM TrisHCI pH 7.4) was performed for 4 min. The reaction was stopped in Tris-HCl pH 8.0, the slides were air-dried, dipped briefly in xylene and coverslipped with Entellan (Merck, Darmstadt, FRG). Neutral red staining for nuclei. The sections were counterstained after in situ hybridization with Neutral red solution (Sigma, St. Louis, USA), air-dried, dipped briefly in xylene and coverslipped with Entellan. A strong hybridization signal was demonstrated with the AT 1 cRNA probe in the SFO. The hybridized material had the appearance of large granules within the cytoplasm of rounded cells, probably representing nerve cells in view of their size, shape and distribution (Fig. la). The control hybridizations with the DIG-11-UTP-labelled AT 1 mRNA probe showed no labelling above the background (Fig. lb). The neuronal and/or glial localization of the hybridized material was also investigated by combining non-radioactive ISH with immunocytochemistry for GFAP. The specifically hybridized dark blue material was found not to be related to the GFAP-IR cells and their processes shown to exist in the SFO (Fig. la,f). The transcription of the AT 1 gene in neuronal cells was further indicated by staining the nuclei with Neutral red after ISH. In these sections, the hybridized material seen with the non-radioactively labelled AT 1 cRNA probe was associated with the large nerve cell nuclei but not with the small glial nuclei (Fig. lc,e). It was found that the specific AT 1 hybridization signal appeared as small to large granules in GFAP-IR-negative putative nerve cells with a perinuclear localization (Fig. If). The strong hybridization signal demonstrated with the angiotensinogen cRNA but not the corresponding mRNA probe in the SFO was located in the astroglial cells, since the hybridized material (dark blue) was present in the cytoplasm of the GFAP-immunoreactive astrocytes (brownish) (Fig. ld). By combining non-radioactive in situ hybridization and GFAP immunocytochemistry or Neutral red staining for nuclei we were able to obtain indications for a neuronal localization of the AT 1 mRNA and to demonstrate angiotensinogen mRNA in astroglial cells in the SFO of the adult rat brain. The specificity of the hybridized material in the SFO was demonstrated by its absence after incubation with the corresponding labelled mRNA probe for the AT 1 receptor as well as for the angiotensinogen. The demonstration of AT 1 mRNA in the SFO using the non-radioactive in situ hybridization
was in good agreement with our earlier results using radioactive in situ hybridization [4]. From angiotensin II receptor binding studies involving receptor subtype characterization, it was also demonstrated that the very strong binding signal detected in the SFO is linked to the AT 1 receptor [9, 25]. Also in the other areas of the brain related to dipsogenic, cardiovascular and endocrine functions, the AT 1 receptor subtype predominates [9, 25, 31]. However, in neuronal cultures the predominating angiotensin II receptor is of the AT 2 type while in cultured astrocytes the AT 1 receptor appears to dominate [27, 28]. These findings are in contrast to our demonstration of AT 1 receptor mRNA in putative neuronal cells in the SFO of the rat brain. One explanation may be the use of primary cultures derived from 1-dayold or 2-week-old rats [26, 28], since a development-dependent change in the expression of the two receptor subtypes may take place [31]. These findings as well as the demonstration of increased expression of AT 2 receptor in the brain of young rats [31] and the demonstration of only low amounts of AT 1 sites in cultured neonatal neurons [28] might suggest a growth and developmentalrelated regulation of the angiotensin II receptor subtype expression in neuronal cells. The demonstration of a predominant neuronal localization of the AT 1 receptor mRNA in the SFO with angiotensinogen mRNA mainly found in the astrocytes of the SFO, supports the proposed major role of volume transmission in the central renin angiotensin system [1, 2]. In previous papers we and others have shown that the precursor peptide of All, angiotensinogen, is transcribed predominantly in the astroglial cells [3, 5, 6], whereas the angiotensin II itself is mainly found in neuronal cells and nerve fibers [3, 6, 17]. The SFO is an organ located outside of the bloodbrain barrier and represents one site of action of circulating All. It may be stimulated by blood born All but also gives rise to AII-immunoreactive projections to the median preoptic nucleus and the paraventricular nucleus of the hypothalamus and receives an All-positive neuronal input from the lateral hypothalamic area [17, 18]. Moreover predominately astroglial cells of the SFO produce angiotensinogen. The question arises of the role of the SFO in linking the peripheral and the central renin angiotensin systems to one another. A major implication of the present paper is that mainly the AT 1 receptor producing SFO nerve cells may be direct targets for circulating All. Thus, All can bind to the neuronal AT 1 receptor of the SFO and induce excitatory actions on the All-positive neuronal output to the hypothalamic neurosecretory neurons [13]. The All in this subfornical output system may act as neurotransmitter in the preoptic region and the paraventricular hypothalamic nucleus
157 ( P V N ) [17-19] to c o n t r o l b l o o d p r e s s u r e a n d b o d y fluid h o m e o s t a s i s b y releasing c o r t i c o t r o p i n - r e l e a s i n g f a c t o r a n d v a s o p r e s s i n a n d to induce d r i n k i n g b e h a v i o r [10, 11, 21, 29]. W i t h r e g a r d to n e u r o n a l f e e d b a c k s to the S F O T a n a k a et al. [30] suggested t h a t the A l l p r o j e c t i o n s to the S F O f r o m the l a t e r a l h y p o t h a l a m i c a r e a c o u l d h a v e an e x c i t a t o r y influence o n the activity o f n e u r o n s in regions o f the S F O with efferent p r o j e c t i o n s to the P V N [18, 30]. T h e l a t e r a l h y p o t h a l a m i c a r e a p l a y s a role in ingestive b e h a v i o r i n c l u d i n g a n g i o t e n s i n - i n d u c e d thirst [6]. T h e s e A I I i n p u t n e u r o n s m a y t h e r e f o r e t r a n s f e r relev a n t i n f o r m a t i o n to the A T 1-positive S F O neurons. It m a y d o so b y c o n t r o l l i n g the b i n d i n g c h a r a c t e r i s t i c s a n d t r a n s d u c t i o n o f the n e u r o n a l A T 1 r e c e p t o r in the S F O . T h e high c o n c e n t r a t i o n s o f n e u r o n a l l y released A l l m a y desensitize the A T 1 r e c e p t o r o f the S F O n e u r o n s , so t h a t c i r c u l a t i n g A l l p r e s e n t in lower c o n c e n t r a t i o n s n o l o n g e r c a n effectively activate the high affinity A T 1 r e c e p t o r o f the S F O . This results in a r e d u c t i o n in the a b i l i t y o f circ u l a t i n g A l l to increase v a s o p r e s s i n a n d c o r t i c o t r o p i n releasing f a c t o r release [15, 21], a r t e r i a l b l o o d p r e s s u r e [12], a n d to induce thirst [14], m a k i n g p o s s i b l e a negative f e e d b a c k r e g u l a t i o n o f the b l o o d - b o r n A l l signal b y the n e u r o n a l A l l like signal. This s t u d y was s u p p o r t e d b y g r a n t s o f the Swedish M e d i c a l R e s e a r c h C o u n c i l (04X715), the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t ( S F B 317) a n d N I H G r a n t 1 RO1 H L 35821-01. 1 Agnati, L.F., Fuxe, K., Zoli, M., Zini, J., Toffano, G. and Ferraguti, F., A correlation analysis of the regional distribution of central enkephalin and fl-endorphin immunoreactive terminals and of opiate receptors in adult and old male rats. Evidence for the existence of two main types of communication in the central nervous system: the volume transmission and the wiring transmission, Acta Physiol. Scand., 128 (1986) 201-207. 2 Bunnemann, B., Fuxe, K., Bjelke, B. and Ganten, D., The brain renin-angiotensin system and its possible involvement in volume transmission. In K. Fuxe and L.F. Agnati (Eds.), Advances in Neuroscience, Vol. 1, Raven, New York, 1990, pp. 131-158. 3 Bunnemann, B., Fuxe, K., Metzger, R., Bjelke, B. and Ganten, D., The semiquantitative distribution and cellular localization of angiotensinogen mRNA in the rat brain, J. Chem. Neuroanatom., 5 (1992) 245-262. 4 Bunnemann, B., Iwai, N., Metzger, R., Fuxe, K., Inagami, T. and Ganten, D., The distribution of angiotensin II AT 1 receptor subtype mRNA in the rat brain, Neurosci. Lett., 140 (1992) 155-158. 5 Deschepper, C.F., Bouhnik, J. and Ganong, W.F., Localization of angiotensinogen and glial fibrillary acidic protein in astrocytes in rat brain, Brain Res., 374 (1986) 195-198. 6 Fuxe, K., Bunnemann, B., Aronsson, M., Tinner, B., Cintra, A., von Euler, G., Agnati, L.F., Nakanishi, S., Ohkubo, H. and Ganten, D., Pre- and postsynaptic features of the central angiotensin systems. Indications for a role of angiotensin peptides in volume transmission and for interactions with central monoamine neurons, Clin. Exp. Hyp. Theory Pract. A, 10 (Suppl. 1) (1988) 143-168.
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