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Localization of angiotensinogen in multiple cell types of rat brain
74
Brain Research, 410 (1987) 74-77 Elsevier
BRE 22169
Localization of angiotensinogen in multiple cell types of rat brain Hans Imboden 1, Joseph W. Harding 2, Ulrich Hilgenfeldt 3, M a r c o R. Celio 4 a n d D o m i n i k Felix 1 ~Division of Animal Physiology, University of Berne, Berne (Switzerland), 2Department VCA PP, Washington State University, Pullman, Washington (U.S.A.), 3Department of Pharmacology, University of Heidelberg, Heidelberg (F. R. G.) and 4Department of Anatomy, University of Zurich, Zurich (Switzerland) (Accepted 16 December 1986) Key words: Angiotensinogen; Angiotensin II; Brain; Astrocyte; Neuron; Choroid plexus; Immunohistochemistry; Rat
Angiotensinogen was localized in 3 cell types in brain using immunohistochemicalmethods. These locations included (1) subpopulations of neurons in nuclei that co-stain for angiotensin II, (2) subpopulations of astrocytes that make putative contacts with brain microvessels, and (3) cells of the choroid plexus. These findings are consistent with multiple functions for brain angiotensinogen as a precursor for neuronal angiotensin II and as a potential source for angiotensin II that is locally produced in the brain. The brain-renin-angiotensin system is considered to be totally endogenous to the brain. Each of its components including: angiotensinogen 1"9, renin 5'6, converting enzyme 6, and angiotensin II (All) itself7, have been identified in brain. Although the presence of all of the components in the brain seems indisputable, the exact cellular location of several key components, including angiotensinogen 2 have been disputed. While All appears to be exclusively located in neurons 8'1° angiotensinogen has been reported to reside only in astrocytes and ependymal cells 2. This apparent paradoxical localization of precursor and product in different cell types necessitates a reevaluation of our current concepts concerning the brain-renin-angiotensin system. As part of this reevaluation, we reexamined the cellular location of angiotensinogen immunoreactivity in rat brain sections at the light microscope level. One gram of CH-Sepharose 4B (Pharmacia) was suspended in 0.5 M NaCI. After a 0.5 M NaC1 and H20 wash, 5 mg of renin substrate tetradecapeptide (Sigma) was added in 2 ml H 2 0 along with 5.5 ml of carbodiimide yielding a total volume of 10.5 ml. After 24 h incubation at room temperature, the gel was successively washed with several cycles of 0.1 M ace-
tate, pH 4, and 0.1 M Tris, pH 8, both containing 1.0 M NaCI. Following additional water and Tris-buffered saline (TBS; 10 mM Tris, pH 7.6) washes, the redissolved pellet resulting from the (NH4)2SO 4 precipitation of 200 t~I of rabbit angiotensinogen antiserum was added. After adding enough TBS to reach a total of 10.5 ml, the suspension was incubated at room temperature for 4 h. The gel was loaded into a glass column and successively eluted with 50 ml of 10 mM Tris, pH 7.6; 20 ml of 10 mM Tris, pH 7.6 with 1.0 M NaC1; 30 ml of 10 mM Tris, pH 7.6; and, 20 ml of 0.1 M glycine HC1, pH 2.8. The purified antibody eluted with this last buffer. Male WKY rats (weight 215 g) were anesthetized and perfused first with 100 ml of heparinized Ringer and then 200 ml of Zamboni's fixative 12. After removal, the brain was postfixed for an additional 24 h in Zamboni's. The brain was then washed and incubated overnight in phosphate-buffered saline (PBS) containing sucrose. The brain was blocked and cut into 30/~m sections. The sections were incubated with affinity-purified angiotensinogen or AII antibody in TBS with 0.1% Triton X-100 at 4 °C for 44 h. After several washes, the sections were incubated with goat anti-rabbit immunoglobulin (dilution 1:30)
Correspondence: D. Felix, Division of Animal Physiology, University of Berne, Erlachstrasse 9a, CH-3012 Berne, Switzerland 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
75 at room temperature for 45 min. Following additional washes the tissue was incubated with peroxidaseantiperoxidase complex in the dark at room temperature for 45 min. Finally the sections were incubated with diaminobenzidine (DAB, 4 mg/10 ml) and 0.3% H202 at room temperature for 15 min. Sections were counterstained with Toluidine blue. Preabsorption controls were produced with the purified antibody when it was first incubated in a batch procedure with the renin substrate tetradecapeptide, that was covalently linked through its N-terminal to CH-sepharose. Incubation with the CH-sepharose alone had no effect on the subsequent staining. Further controls, in which preimmune serum or no first antibody had been applied, also exhibited no staining. Our immunohistochemical study revealed that a large portion of the total angiotensinogen immunoreactivity in brain was present in astrocytes. It appeared that only a subpopulation of astrocytes contained angiotensinogen since almost no staining was seen dorsal to the corpus callosum. The highest concentration of astrocytic staining was evident in the arcuate nucleus. Of potential importance is the tendency of angiotensinogen-containing astrocytes to make contact with brain microvessels. Such an astrocyte can be seen in Fig. IA. For comparison's sake another astrocyte, which is stained for glial fibrillary acidic protein (GFAP), is seen making intimate contact with vessels (Fig. 1B). Astrocytes did not stain for angiotensin II. Although most of the total angiotensinogen staining appears to be astrocytic, clear staining of neurons was evident in paraventricular, supraoptic and accessory magnoceUular nuclei. Interestingly, these same nuclei always co-stained for AII. Fig. 1 (C, D) illustrates an example of the ventral base of the hypothalamus, adjacent to the ventral supraoptic commissure where neurons stained both for angiotensinogen (Fig. 1C) and All (Fig. 1D). Further studies will show whether there exists the same kind of co-staining in nuclei of regions of the brain other than the hypothalamus in which AII staining has been reported. A third cellular location for angiotensinogen was the cells of the choroid plexus (Fig. 1E). These cells were characterized by punctate staining that seemed universal for this cell type. The location of angiotensinogen in 3 distinct cell
types with limited distribution suggests that angiotensinogen may serve several functions in the brain. Its exclusive neuronal localization in nuclei that also stain for AII provides strong support for the presence of an endogenous brain-angiotensin system. The presence of angiotensinogen in a subpopulation of astrocytes that make putative contact with brain microvessels suggests two potential functions. Astrocytic angiotensinogen could represent the precursor pool for a second brain-angiotensin system that is exclusively associated with blood vessels and involved with the regulation of cerebral blood flow. This angiotensinogen could be secreted into the vessel lumen where it could be converted to AII and react with local vascular receptors. A similar vascular wall renin-angiotensin pathway has already been proposed as a possible mechanism for the control of peripheral blood flow3. The basis for his proposal has been the identification of several components of the renin-angiotensin system, including angiotensinogen, in the cells of the vascular wall 4. Although the source of astrocytic angiotensinogen has yet to be identified, the high levels of angiotensinogen message in brain I and the predominance of angiotensinogen in astrocytes 2 suggest that astrocytes themselves may be the site of synthesis. An alternative to the above proposal would involve a unique relationship between angiotensinogen-containing astrocytes and angiotensin-producing neurons. Such a system would require direct contact between these elements so that secreted angiotensinogen could be taken up by the neurons. The close association between the astrocytes and the blood vessels encourages one to further speculate that the alternative source of angiotensinogen may be the plasma and that it is being taken up selectively by the astrocytes. The final location of angiotensinogen is the choroid plexus. The high concentration of angiotensinogen in cerebrospinal fluid (CSF) compared to plasma t' indicates that angiotensinogen is being actively secreted into the CSF. The results of this study suggest two plausible sources: (1) astrocytes, and (2) from the blood via the choroid plexus. In total the results of this study demonstrate the presence of angiotensinogen in 3 cell types in rat brain. This finding is consistent with multiple functions which include the role of angiotensinogen as the
&
•
o
,
77 Fig. 1. lmmunohistochemical localization of angiotensinogen and angiotensin II in rat hypothalamus using the indirect peroxidaseantiperoxidase technique. A: angiotensinogen staining in astrocyte associated with a brain microvessel. ×400. B: glial fibrillary acidic protein (GFAP) staining in astrocytes associated with a microvessel, x576. C, D: two adjacent 301~m sections of the ventral base of the hypothalamus, In C angiotensinogen staining is shown in astrocytes (arrow heads) and neurons (arrows). In D angiotensin II-like immunoreactive products are present in neurons (arrows) of the same area shown in C. Immunoreactive cells can be distinguished from Toluidine blue counterstained, non-reactive cells. Furthermore, immunoreactive fiber pathways of the ventral supraoptic commissure are clearly visible. ×272. E: angiotensinogen staining in choroid plexus. ×800. Bar in A, B, E = 10,um; bar in C, D = 20/~m.
p r e c u r s o r to n e u r o n a l A I I and a p o t e n t i a l r e g u l a t o r
F o u n d a t i o n to D . F . F u r t h e r m o r e , this w o r k was sup-
of c e r e b r a l b l o o d flow.
p o r t e d by financial aid f r o m the ' S t i f t u n g zur F 6 r d e r u n g d e r w i s s e n s c h a f t l i c h e n F o r s c h u n g an der U n i -
This w o r k was s u p p o r t e d G r a n t s T W 01112 and H L 32063 f r o m the N I H
and G r a n t 831145 f r o m the
A m e r i c a n H e a r t A s s o c i a t i o n to J . W . H . ,
as well as
versit~it B e r n ' to H , I , and D . F . W e w o u l d especially like to t h a n k R. Bandi for p r e p a r i n g this m a n u s c r i p t and B. O e s c h for technical assistance.
G r a n t 3.627-0.84 f r o m the Swiss N a t i o n a l S c i e n c e
1 Campbell, D.J., Bouhnik, J., Mevard, J. and Corvol, P., Identity of angiotensinogen precursors of rat brain and liver, Nature (London) 308 (1984) 206-208. 2 Deschepper, C.F., Bouhnik, J. and Ganong, W.F,, Co-localization of angiotensinogen and glial fibrillary acidic protein in astrocytes in rat brain, Brain Research, 355 (1986) 195-198. 3 Dzau, V.J., Vascular wall renin-angiotensin pathway in control of the circulation, Am. J. Med.. October 5 (1984) 31-36, 4 Dzau, V.J., Vascular renin-angiotensin: a possible autocrine or paracrine system in control of vascular function, J. Cardiovasc, Pharmacol., 6 (1984) $377-$382. 5 Ganten, D. and Speck, G., The brain-renin-angiotensin system, a model for the synthesis of peptides in the brain, Biochem. Pharrnacol., 27 (1978) 2379-2389, 6 Ganten, D., Lang, R.E,, Lehmann, E, and Unger, T., Brain angiotensin: on the way to becoming a well-studied neuropeptide system, Biochem. Pharmacol., 33 (1984) 3523-3528, 7 Hermann, K,, McDonald, W., Unger, T., Lang, R,E. and
Ganten, D.. Angiotensin biosynthesis and concentrations in brain of normotensive and hypertensive rats, J. Physiol. (Paris), 79 (1984) 471-480. 8 Imboden, H., Harding, J. and Felix, D., Comparison of immunostaining patterns in rat brain using crude and affinity purified antisera for angiotensin II and angiotensinogen, Abstract S. 323, 10th Annual Meeting of the European Neuroscience Association, Marseille, 1986. 9 Lewicki, J.A., Fallen, J.H. and Printz, M.P., Regional distribution of angiotensinogen in rat brain, Brain Research. 158 (1978) 359-371. 10 Lind, R.W., Swanson, L.W. and Ganten, D., Organization of angiotensin II immunoreactive cells and fibers in rat central nervous system, An immunohistochemicat study, Neuroendocrinology, 40 (1985) 2-24. t 1 Schelling, P., Clauser, E. and Felix, D., Regulation of angiotensinogen in the central nervous system, Clin. Exp. Hy, p e r . - Theory' and Practice, A5 (1983) 1047-106t. 12 Zamboni, L. and De Martino, C.. Buffered picric-acid formaldehyde: a new, rapid fixative for electron microscopy, J. Cell Biol.. 35 (1967) 148A.
Brain Research, 410 (1987) 74-77 Elsevier
BRE 22169
Localization of angiotensinogen in multiple cell types of rat brain Hans Imboden 1, Joseph W. Harding 2, Ulrich Hilgenfeldt 3, M a r c o R. Celio 4 a n d D o m i n i k Felix 1 ~Division of Animal Physiology, University of Berne, Berne (Switzerland), 2Department VCA PP, Washington State University, Pullman, Washington (U.S.A.), 3Department of Pharmacology, University of Heidelberg, Heidelberg (F. R. G.) and 4Department of Anatomy, University of Zurich, Zurich (Switzerland) (Accepted 16 December 1986) Key words: Angiotensinogen; Angiotensin II; Brain; Astrocyte; Neuron; Choroid plexus; Immunohistochemistry; Rat
Angiotensinogen was localized in 3 cell types in brain using immunohistochemicalmethods. These locations included (1) subpopulations of neurons in nuclei that co-stain for angiotensin II, (2) subpopulations of astrocytes that make putative contacts with brain microvessels, and (3) cells of the choroid plexus. These findings are consistent with multiple functions for brain angiotensinogen as a precursor for neuronal angiotensin II and as a potential source for angiotensin II that is locally produced in the brain. The brain-renin-angiotensin system is considered to be totally endogenous to the brain. Each of its components including: angiotensinogen 1"9, renin 5'6, converting enzyme 6, and angiotensin II (All) itself7, have been identified in brain. Although the presence of all of the components in the brain seems indisputable, the exact cellular location of several key components, including angiotensinogen 2 have been disputed. While All appears to be exclusively located in neurons 8'1° angiotensinogen has been reported to reside only in astrocytes and ependymal cells 2. This apparent paradoxical localization of precursor and product in different cell types necessitates a reevaluation of our current concepts concerning the brain-renin-angiotensin system. As part of this reevaluation, we reexamined the cellular location of angiotensinogen immunoreactivity in rat brain sections at the light microscope level. One gram of CH-Sepharose 4B (Pharmacia) was suspended in 0.5 M NaCI. After a 0.5 M NaC1 and H20 wash, 5 mg of renin substrate tetradecapeptide (Sigma) was added in 2 ml H 2 0 along with 5.5 ml of carbodiimide yielding a total volume of 10.5 ml. After 24 h incubation at room temperature, the gel was successively washed with several cycles of 0.1 M ace-
tate, pH 4, and 0.1 M Tris, pH 8, both containing 1.0 M NaCI. Following additional water and Tris-buffered saline (TBS; 10 mM Tris, pH 7.6) washes, the redissolved pellet resulting from the (NH4)2SO 4 precipitation of 200 t~I of rabbit angiotensinogen antiserum was added. After adding enough TBS to reach a total of 10.5 ml, the suspension was incubated at room temperature for 4 h. The gel was loaded into a glass column and successively eluted with 50 ml of 10 mM Tris, pH 7.6; 20 ml of 10 mM Tris, pH 7.6 with 1.0 M NaC1; 30 ml of 10 mM Tris, pH 7.6; and, 20 ml of 0.1 M glycine HC1, pH 2.8. The purified antibody eluted with this last buffer. Male WKY rats (weight 215 g) were anesthetized and perfused first with 100 ml of heparinized Ringer and then 200 ml of Zamboni's fixative 12. After removal, the brain was postfixed for an additional 24 h in Zamboni's. The brain was then washed and incubated overnight in phosphate-buffered saline (PBS) containing sucrose. The brain was blocked and cut into 30/~m sections. The sections were incubated with affinity-purified angiotensinogen or AII antibody in TBS with 0.1% Triton X-100 at 4 °C for 44 h. After several washes, the sections were incubated with goat anti-rabbit immunoglobulin (dilution 1:30)
Correspondence: D. Felix, Division of Animal Physiology, University of Berne, Erlachstrasse 9a, CH-3012 Berne, Switzerland 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
75 at room temperature for 45 min. Following additional washes the tissue was incubated with peroxidaseantiperoxidase complex in the dark at room temperature for 45 min. Finally the sections were incubated with diaminobenzidine (DAB, 4 mg/10 ml) and 0.3% H202 at room temperature for 15 min. Sections were counterstained with Toluidine blue. Preabsorption controls were produced with the purified antibody when it was first incubated in a batch procedure with the renin substrate tetradecapeptide, that was covalently linked through its N-terminal to CH-sepharose. Incubation with the CH-sepharose alone had no effect on the subsequent staining. Further controls, in which preimmune serum or no first antibody had been applied, also exhibited no staining. Our immunohistochemical study revealed that a large portion of the total angiotensinogen immunoreactivity in brain was present in astrocytes. It appeared that only a subpopulation of astrocytes contained angiotensinogen since almost no staining was seen dorsal to the corpus callosum. The highest concentration of astrocytic staining was evident in the arcuate nucleus. Of potential importance is the tendency of angiotensinogen-containing astrocytes to make contact with brain microvessels. Such an astrocyte can be seen in Fig. IA. For comparison's sake another astrocyte, which is stained for glial fibrillary acidic protein (GFAP), is seen making intimate contact with vessels (Fig. 1B). Astrocytes did not stain for angiotensin II. Although most of the total angiotensinogen staining appears to be astrocytic, clear staining of neurons was evident in paraventricular, supraoptic and accessory magnoceUular nuclei. Interestingly, these same nuclei always co-stained for AII. Fig. 1 (C, D) illustrates an example of the ventral base of the hypothalamus, adjacent to the ventral supraoptic commissure where neurons stained both for angiotensinogen (Fig. 1C) and All (Fig. 1D). Further studies will show whether there exists the same kind of co-staining in nuclei of regions of the brain other than the hypothalamus in which AII staining has been reported. A third cellular location for angiotensinogen was the cells of the choroid plexus (Fig. 1E). These cells were characterized by punctate staining that seemed universal for this cell type. The location of angiotensinogen in 3 distinct cell
types with limited distribution suggests that angiotensinogen may serve several functions in the brain. Its exclusive neuronal localization in nuclei that also stain for AII provides strong support for the presence of an endogenous brain-angiotensin system. The presence of angiotensinogen in a subpopulation of astrocytes that make putative contact with brain microvessels suggests two potential functions. Astrocytic angiotensinogen could represent the precursor pool for a second brain-angiotensin system that is exclusively associated with blood vessels and involved with the regulation of cerebral blood flow. This angiotensinogen could be secreted into the vessel lumen where it could be converted to AII and react with local vascular receptors. A similar vascular wall renin-angiotensin pathway has already been proposed as a possible mechanism for the control of peripheral blood flow3. The basis for his proposal has been the identification of several components of the renin-angiotensin system, including angiotensinogen, in the cells of the vascular wall 4. Although the source of astrocytic angiotensinogen has yet to be identified, the high levels of angiotensinogen message in brain I and the predominance of angiotensinogen in astrocytes 2 suggest that astrocytes themselves may be the site of synthesis. An alternative to the above proposal would involve a unique relationship between angiotensinogen-containing astrocytes and angiotensin-producing neurons. Such a system would require direct contact between these elements so that secreted angiotensinogen could be taken up by the neurons. The close association between the astrocytes and the blood vessels encourages one to further speculate that the alternative source of angiotensinogen may be the plasma and that it is being taken up selectively by the astrocytes. The final location of angiotensinogen is the choroid plexus. The high concentration of angiotensinogen in cerebrospinal fluid (CSF) compared to plasma t' indicates that angiotensinogen is being actively secreted into the CSF. The results of this study suggest two plausible sources: (1) astrocytes, and (2) from the blood via the choroid plexus. In total the results of this study demonstrate the presence of angiotensinogen in 3 cell types in rat brain. This finding is consistent with multiple functions which include the role of angiotensinogen as the
&
•
o
,
77 Fig. 1. lmmunohistochemical localization of angiotensinogen and angiotensin II in rat hypothalamus using the indirect peroxidaseantiperoxidase technique. A: angiotensinogen staining in astrocyte associated with a brain microvessel. ×400. B: glial fibrillary acidic protein (GFAP) staining in astrocytes associated with a microvessel, x576. C, D: two adjacent 301~m sections of the ventral base of the hypothalamus, In C angiotensinogen staining is shown in astrocytes (arrow heads) and neurons (arrows). In D angiotensin II-like immunoreactive products are present in neurons (arrows) of the same area shown in C. Immunoreactive cells can be distinguished from Toluidine blue counterstained, non-reactive cells. Furthermore, immunoreactive fiber pathways of the ventral supraoptic commissure are clearly visible. ×272. E: angiotensinogen staining in choroid plexus. ×800. Bar in A, B, E = 10,um; bar in C, D = 20/~m.
p r e c u r s o r to n e u r o n a l A I I and a p o t e n t i a l r e g u l a t o r
F o u n d a t i o n to D . F . F u r t h e r m o r e , this w o r k was sup-
of c e r e b r a l b l o o d flow.
p o r t e d by financial aid f r o m the ' S t i f t u n g zur F 6 r d e r u n g d e r w i s s e n s c h a f t l i c h e n F o r s c h u n g an der U n i -
This w o r k was s u p p o r t e d G r a n t s T W 01112 and H L 32063 f r o m the N I H
and G r a n t 831145 f r o m the
A m e r i c a n H e a r t A s s o c i a t i o n to J . W . H . ,
as well as
versit~it B e r n ' to H , I , and D . F . W e w o u l d especially like to t h a n k R. Bandi for p r e p a r i n g this m a n u s c r i p t and B. O e s c h for technical assistance.
G r a n t 3.627-0.84 f r o m the Swiss N a t i o n a l S c i e n c e
1 Campbell, D.J., Bouhnik, J., Mevard, J. and Corvol, P., Identity of angiotensinogen precursors of rat brain and liver, Nature (London) 308 (1984) 206-208. 2 Deschepper, C.F., Bouhnik, J. and Ganong, W.F,, Co-localization of angiotensinogen and glial fibrillary acidic protein in astrocytes in rat brain, Brain Research, 355 (1986) 195-198. 3 Dzau, V.J., Vascular wall renin-angiotensin pathway in control of the circulation, Am. J. Med.. October 5 (1984) 31-36, 4 Dzau, V.J., Vascular renin-angiotensin: a possible autocrine or paracrine system in control of vascular function, J. Cardiovasc, Pharmacol., 6 (1984) $377-$382. 5 Ganten, D. and Speck, G., The brain-renin-angiotensin system, a model for the synthesis of peptides in the brain, Biochem. Pharrnacol., 27 (1978) 2379-2389, 6 Ganten, D., Lang, R.E,, Lehmann, E, and Unger, T., Brain angiotensin: on the way to becoming a well-studied neuropeptide system, Biochem. Pharmacol., 33 (1984) 3523-3528, 7 Hermann, K,, McDonald, W., Unger, T., Lang, R,E. and
Ganten, D.. Angiotensin biosynthesis and concentrations in brain of normotensive and hypertensive rats, J. Physiol. (Paris), 79 (1984) 471-480. 8 Imboden, H., Harding, J. and Felix, D., Comparison of immunostaining patterns in rat brain using crude and affinity purified antisera for angiotensin II and angiotensinogen, Abstract S. 323, 10th Annual Meeting of the European Neuroscience Association, Marseille, 1986. 9 Lewicki, J.A., Fallen, J.H. and Printz, M.P., Regional distribution of angiotensinogen in rat brain, Brain Research. 158 (1978) 359-371. 10 Lind, R.W., Swanson, L.W. and Ganten, D., Organization of angiotensin II immunoreactive cells and fibers in rat central nervous system, An immunohistochemicat study, Neuroendocrinology, 40 (1985) 2-24. t 1 Schelling, P., Clauser, E. and Felix, D., Regulation of angiotensinogen in the central nervous system, Clin. Exp. Hy, p e r . - Theory' and Practice, A5 (1983) 1047-106t. 12 Zamboni, L. and De Martino, C.. Buffered picric-acid formaldehyde: a new, rapid fixative for electron microscopy, J. Cell Biol.. 35 (1967) 148A.