Immunohistochemical demonstration of angiotensin II receptors in rat brain by use of an anti-idiotypic antibody

Regulatory Peptides, 44 (1993) 109-117

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Immunohistochemical demonstration of angiotensin II receptors in rat brain by use of an anti-idiotypic antibody JOrg Pfister, D o m i n i k Felix and H a n s I m b o d e n University of Berne, Division of Neurobiology, Berne (Switzerland)

(Received 6 October 1992; accepted 11 December 1992) K e y words: Immunohistochemistry; Angiotensin receptor; Anti-idiotypic antibody; Rat; Supraoptic nucleus;

Paraventricular nucleus

Summary In the present study we investigated the ability of an anti-idiotypic antibody which recognizes angiotensin II (AII) receptors to demonstrate the presence of such receptors under immunohistochemical conditions. The experiments revealed punctate immunoreactive granules on neurons of the nucleus supraopticus and of the nucleus paraventricularis of the hypothalamus. This localization of AII receptors is consistent with the findings obtained using other experimental approaches to the brain renin-angiotensin system. The conclusion of this study is that the applied anti-idiotypic antibody seems to be a reliable tool for mapping AII receptor distribution. The established experimental approaches to AII receptors are thus now supplemented with the possibility of immunohistochemical investigation. Moreover, the possible microscopic analysis of AII receptors on distinct cells will allow studies at an ultrastructural level.

Introduction The description of idiotypes [ 1,2] provided the basis for the formulation of the network theory of the immune system [3 ]. One consequence of this theory and of the internal image concept was that one should be able to demonstrate anti-idiotypic (anti-Id) antiCorrespondence to: H. Imboden, Universityof Berne, Divisionof Neurobiology,Erlachstrasse 9a, 3012 Berne, Switzerland.

bodies that recognize receptors. This was first achieved by Sege and Peterson in the insulin and retinol systems [4]. Since then numerous other authors have characterized anti-Id antibodies in various systems (for review see Ref. 5). In this study we worked with a polyclonal anti-Id antibody which had earlier been shown to react towards the angiotensin II (All) receptors of rat liver membranes. A number of experiments had revealed that this antibody reacts towards a public idiotope,

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111 located outside the hormone binding site [6]. The aim of our study was to establish this antibody's capacity to demonstrate the presence of AII receptors in the rat brain under immunohistochemical conditions. For this purpose we chose to examine the nucleus supraopticus (SON) and the nucleus paraventricularis (PVN) of the hypothalamus. These hypothalamic areas play an important role in the functioning of the brain renin-angiotensin system (RAS) [7]. In the PVN [8-12] as well as in the SON [ 12-14] the presence of AII receptors has been demonstrated using autoradiography and electrophysiology. So far, however, no immunohistochemical approach to AII receptors using anti-Id antibodies has been reported.

Materials and Methods Anti-idiotypic serum The anti-Id serum was prepared by P.O. Couraud at the Centre C N R S - I N S E R M de PharmacologieEndocrinologie, Montpellier, France, and is characterized as follows [6]: (i) The xenogenic anti-Id serum (R anti-A25) from a rabbit (R) recognized a private idiotope located near the hormone binding site, on the anti-AII monoclonal antibody (A25) against which it had been produced. (ii) R anti-A25 recognized a crossreactive idiotope on six monoclonal anti-All antibodies. This public idiotope seems to be located outside the hormone binding site. The anti-Id antibodies that recognized it seem to be clearly different from internal images of AII. (iii) R anti-A25 bound to rat liver membranes which bear AII receptors. (iv) In immu-

noblotting assays with rat liver membranes as well as rat anterior pituitary homogenates which bear AII receptors, the anti-Id R anti-A25 serum revealed one protein with the same 63,000 + 2000 D a molecular mass of the AII receptor determined after photoaffinity labelling. (v) The R anti-A25 serum was separated on an affinity column into a fraction containing the anti-Id antibodies directed towards the private, binding-site-related idiotope and a fraction containing the anti-Id antibodies which recognize the public idiotope located outside the hormone binding site. After this separation only the latter fraction identified the 63,000 D a band by immunoblotting, but it did not compete with the hormone for binding to A25. This fraction was used in the present study. Anti-angiotensin H serum The specific anti-All antibody ' B O D E ' used in this study is described in detail elsewhere [15]. A rabbit had been immunized with a haptencarrier-conjugate consisting of IleS-AII and bovine serum albumin. The resulting polyclonal anti-All antiserum was then purified on a CH-sepharose 4B affinity column with AII bound to the resin [16]. Immunohistochemistry Male Wistar Kyoto rats (WKY) between 19 and 27 weeks of age and having an average weight of 430 g were used in this study. The rats were anesthetized with a 5 % solution of thiopentane sodium (300 #1/ 100 g body weight, i.p.). The chest was opened and the descending aorta was clamped. After an incision had been made into the right atrium, 200 ml of Ringer solution containing 2000 U heparin and 2 g MgC12 x 6 H 2 0 at 37°C were injected into the left

Fig. 1. (A) Light-microscopicvisualization of immunoreactiveAll-containing neurons (black cells) and fibers in the SON of the rat hypothalamus. Analysis of all such preparations showed a tendency of these neurons to be concentrated in the ventral part of the SON. The grey cells do not contain AII; their coloration is due to the counterstaining with Toluidine blue. (B) Demonstration of immunoreactiveAII receptors using an anti-idiotypic antibody in the section adjacent to A. The micrograph demonstrates that the AII-receptor-bearingneurons are distributed regularly over the entire nucleus. (Bl) High-magnificationmicrograph of B (area marked with arrow-head) demonstrating immunoreactive AII receptors on neurons of the SON. Some granules are also localized on cellular processings. TO = tractus opticus. Scale bars: 50 #m in (A) and (B), 10/~m in (Bl).

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113 ventricle under 75 cm hydrostatic pressure. This was immediately followed by 200 ml of fixative (2~o paraformaldehyde in phosphate buffer, p H 7.3) at 4°C, which required about 20 min. The brain was carefully removed and cut into appropriate pieces. These were further incubated for 4 h in 50 ml of fixative, which was subsequently replaced with 50 ml of fresh fixative and the postfixation was continued for a further 24 h. The brain tissue was then washed overnight in 100 ml of phosphate-buffered saline (PBS-Dulbecco) containing 18 g of sucrose and 50 mg N a N 3. The brain pieces were fixed to the cryostat chucks by CO 2 freezing and cut into 30/zm sections which were then immediately transferred to PBS at room temperature. After a 15-min wash in PB S and two 15-min washes in TBS-Triton (5 m M Tris at p H 7.6 containing 0.1 ~ Triton X-100) the staining procedure was begun. The tissue was incubated for 44 h at 4 ° C with continuous shaking in a solution containing either the anti-Id antibodies or the anti-AII antibodies in TBS-Triton plus 0.05~o N a N 3. After the first incubation the sections were washed three times for 15 min in TBS-Triton. The sections were subsequently incubated with a 1:30 dilution of goat-anti-rabbit immunoglobulins (Nordic Immunological Laboratories, Tilburg, Netherlands) for 1 h at room temperature. This was followed by three TB S-Triton washes of 15 min each. After having been washed the tissue was incubated with the rabbit peroxidase-antiperoxidase complex (Sternberger-Meyer Immunocytochemicals, Jarretsville, PA, U S A ) at a 1:200 dilution for 1 h at room temperature. The sections were again washed for 15 min in TBS-Triton and twice in TBS only. They were then incubated for 10 min at room temperature in a solution containing 2.8 ml of

50 m M Tris (pH 7.6), 140 #1 of the 3,3'-diaminobenzidine tetrahydrochloride concentrate (50 mg/5 ml H20; Polyscience) and 28/~1 of 0.3~o H202. Finally, the sections were washed twice more with TB S at room temperature for 15 min. The stained sections were then picked up on gelatin-coated slides, dried and counterstained with Toluidine blue.

Results In the present study we investigated the ability of an anti-Id antibody which recognizes AII receptors to demonstrate such receptors under immunohistochemical conditions. The revealed immunoreactive staining in coronal sections of the rat hypothalamus was compared with that yielded by a polyclonal, affinity-purified anti-AII antibody in adjacent sections. In the S O N (Fig. 1) AII-containing neurons which were stained regularly all over their cell bodies tended to be concentrated in the ventral part of the nucleus (Fig. 1A). Fine varicose fibers were stained as well. The approach with the anti-Id antibody to AII receptors revealed punctate immunoreactive granules on the cells in the area examined. In the S O N these granules were especially abundant, most of the supraoptic magnocellular neurons being covered with them (Fig. 1B). Their regular distribution over the entire nucleus contrasts to the ventral concentration of AII-containing neurons. The PVN (Fig. 2), the second nucleus in the examination area, revealed the same punctate granules as the SON. The neurons bearing these AII receptors were regularly and densely distributed mainly in its magnocellular parts. Comparative analysis of the

Fig. 2. (A) Light-microscopicvisualization of immunoreactiveAll-containing neurons (black cells) and fibers in the PVN of the rat hypothalamus. The grey cells do not contain All; their coloration is due to the counterstaining with Toluidine blue. (B) Demonstration of immunoreactiveAII receptors using an anti-idiotypicantibodyin the section adjacent to A. The AII-receptor-bearingneurons are distributed regularly over the magnocellularpart. (B~) High-magnificationmicrograph of B (area marked with arrow-head) demonstrating immunoreactive AII receptors on neurons of the PVN. III = 3rd ventricle. Scale bars: 50/am in (A) and (B), 10/~m in (B1).

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Fig. 3. Light-microscopicvisualization of immunoreactive AII receptors using an anti-idiotypic antibody on a cell of the ependymal layer of the third ventricle near the PVN in the rat hypothalamus. III = 3rd ventricle. Scale bar: 10 #m. presence o f A l l in the neurons o f adjacent sections showed that while in "some areas of the P V N A I I receptors are quite abundant, All-containing neurons are almost absent. In other parts both were found in equal abundance. In this study we focused our attention on the S O N and the PVN. Nevertheless, AII-receptor-bearing cells were also found in other areas. Fig. 3 shows this for cells of the ependymal layer o f the third ventricle and Fig. 4 for the plexus choroideus. All-containing and AII-receptor-bearing cells were also localized in the retrochiasmatic part of the S O N as well as in accessory magnocellular nuclei of the hypothalamus (data not shown).

Fig. 4. Light-microscopicvisualization ofimmunoreactive AII receptors using an anti-idiotypic antibody on cells of the plexus choroideus in the rat brain. Scale bar: 20 #m.

'Omission controls', i.e., incubations without the first antibody (anti-Id or anti-AII antibody), the goatanti-rabbit immunoglobulins or the peroxidase-antiperoxidase complex were carded out and revealed the absence of immunoreactive staining.

Discussion

Antibody specificity For the first time we used an anti-Id antibody which recognizes A I I receptors in an immunohistochemical study of the brain RAS. The specificity of this antibody towards the A I I receptor had been

115 demonstrated with a number of biochemical, nonimmunohistochemical experiments [6]. The application of competition experiments under immunohistochemical conditions (e.g., with tissue fixed in paraformaldehyde) as a control is not suitable in this case. A number of authors have conducted such experiments with different anti-Id antibodies [17-20]. They showed that the anti-Id antibody/receptor interaction is inhibited by the receptor ligand. This experimental approach implies that the antibodies either correspond to Ab2fl anti-Id antibodies, i.e., they exhibit internal image properties [21], or to Ab27 anti-Id antibodies, which can inhibit the binding of the antigen to the paratope of Abl by alteration of the three-dimensional structure of the latter [22]. When such control experiments were conducted under immunohistochemical conditions with AII or saralasin (Sar l, IleS-AII) as competitors the anti-Id antibody/receptor interaction was not inhibited. This was in agreement with the earlier biochemical results where an absence of competition had been estabfished as well [6]. These observations are consistent with the idea that cross-reacting anti-Id antibodies might be directed towards an antigenic determinant that is distinct from the AII binding site, but is always associated with it on AII binding molecules, either antibody or receptor. This structure could be an 'obligatory non-paratopic idiotope' [23], i.e., a structure obligatorily required for the formation of the appropriate antigen-binding site. The antibodies which recognized the AII receptors could therefore correspond to Ab2~ anti-Id antibodies [6]. Until now no convincing control experiment that is directly applicable to fixed tissue slices has been developed for Ab2~ anti-Id antibodies. But in the present case the anti-Id antibody's specificity towards the AII receptor had been demonstrated biochemically [6]. Localization of AH receptors In order to establish the anti-Id antibody's capacity to reveal AII receptors under immunohistochem-

ical conditions, two distinct nuclei were chosen for examination. This study reveals that in the magnocellular parts of the PVN AII receptors seem to be widespread. This supports the findings of other investigations [8-12] and confirms the importance of the PVN for the brain RAS. In addition, the results indicate that in some areas of the PVN All receptors are quite abundant while All-containing neurons are almost absent. Thus, it could be that angiotensinergic fiber-endings influence neurons in the PVN which themselves use angiotensin, other transmitters or neurohormones. In an earlier study we demonstrated the colocalization of AII and vasopressin in neurons as well as in fibers of the hypothalamo-neurohypophysial system [24]. Other studies have revealed that AII affects the firing of paraventricular neuron s [ 9,32 ], some of which may be assumed to be vasopressinergic and cause vasopressin release [25,26]. As the hypothalamohypophysial system originates also, but not exclusively, in the magnocellular parts of the PVN its efferent fibers would seem to be influenced at least to a certain extent by angiotensinergic neurons. Hence, the release of vasopressin and oxytocin in the pituitary depends on the RAS. The RAS probably exerts this influence not only via the PVN but also via the SON [25,27]. The existence of AII-receptor-bearing neurons in the SON has been reported [12-14]. Our immunohistochemical approach revealed that in the rat hypothalamus AII receptors are localized on most of the magnocellular supraoptic neurons. As in the PVN, the distribution pattern of AII receptors and AII does not match exactly, the AII containing perikarya being concentrated ventrally. This must not be considered unusual as a similarly irregular distribution pattern has already been reported for other substances. This accounts for oxytocin's being mapped in supraoptic neurons localized in the dorsal part of the SON and for vasopressin's being found to be concentrated mainly in the ventral cells of the nucleus [28].

116 A I I - r e c e p t o r - b e a r i n g cells were found in other areas a p a r t from the P V N and the S O N , on which the attention of this study was focused. F o r the e p e n d y m a l layer o f the third ventricle a n d the plexus choroideus these results are in agreement with autoradiographic findings [14,29]. The ventricular walls and the circumventricular organs are k n o w n to be o f great i m p o r t a n c e to the central nervous system as their c o n t a c t with the b l o o d is not completely restricted by the b l o o d - b r a i n barrier. O u r d e m o n s t r a tion o f A I I receptors in these areas thus s u p p o r t s the theory that the R A S is involved in i m p o r t a n t functions such as mediating f e e d b a c k messages from the periphery to the brain. T o conclude, the applied anti-Id a n t i b o d y seems to be a reliable tool for m a p p i n g the distribution o f A I I receptors. The established experimental a p p r o a c h e s to A I I receptors are thus s u p p l e m e n t e d with the p o s sibility o f i m m u n o h i s t o c h e m i c a l investigations. M o r e o v e r , the possible m i c r o s c o p i c analysis o f A l l receptors on distinct cells enables studies at an ultrastructural level not k n o w n by a u t o r a d i o g r a p h y . F o r the future it remains to be established whether the i m m u n o r e a c t i v e granules which were revealed really c o r r e s p o n d to synapses where p r e s y n a p t i c endings release A I I to stimulate p o s t s y n a p t i c A l l receptors. A n o t h e r u n a n s w e r e d question is whether the anti-Id a n t i b o d y is directed t o w a r d s a specific A I I r e c e p t o r subtype or not. A n u m b e r o f studies indicate that the A l l receptors in the S O N a n d the P V N [13,14,2932] are o f subtype I. The a n t i b o d y might, however, recognize antigenic d e t e r m i n a n t s that the different receptor subtypes have in c o m m o n .

Acknowledgements W e are especially grateful to P.O. C o u r a u d , J. M a r i e a n d S. J a r d o f the Centre C N R S - I N S E R M de P h a r m a c o l o g i c - Endocrinologie in Montpellier, F r a n c e , w h o supplied us with the anti-idiotypic antibody. This w o r k was s u p p o r t e d by grant No. 3 1 29986.90 (to D . F a n d H . I . ) from the Swiss N a t i o n a l

Science F o u n d a t i o n and by the 'Stiftung zur FOrderung der wissenschaftlichen F o r s c h u n g an der Universitat Bern'. W e wish to t h a n k Mrs. S. G y g a x for technical assistance and Mrs. R. Bandi for preparing the manuscript.

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