(Hypertension. 1996;28:818-824.)
© 1996 American Heart Association, Inc.
Articles |
Subcellular Localization of Angiotensin II Immunoreactivity in the Rat Cerebellar Cortex
the Max-Delbruck-Center for Molecular Medicine, Berlin-Buch, Germany (B.E., D.G.), and the Karolinska Institute, Department of Neuroscience, Division of Cellular and Molecular Neurochemistry, Stockholm, Sweden (K.F.).
Correspondence to B. Erdmann, Max-Delbruck-Center for Molecular Medicine, Electron Microscopy, Robert-Rossle-Str. 10, D-13122 Berlin-Buch, FRG. E-mail berdma@orion.rz.mdc-berlin.de.
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Abstract |
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We localized angiotensin II (Ang II) immunoreactivity in the rat cerebellar cortex with immunogold staining methods. Perfusion fixation with high amounts of glutaraldehyde and the use of cryoultramicrotomy caused remarkable changes in immunostaining versus formaldehyde/picric acid fixation. With the use of monoclonal and polyclonal anti–Ang II, Ang II immunoreactivity was prominent in cerebellar neurons such as Purkinje, granule, basket, and stellate cells. At the subcellular level, the peptide was clearly localized in nuclei, and in some cell types, such as endothelial and granule cells, it was nearly exclusively present in the transcriptionally active euchromatin. Intracellular Ang II immunoreactivity was also detected in vesicle-like structures in cytoplasm and mitochondria and at cell-cell contacts. Additional experiments with liver and adrenal tissue confirmed the nuclear localization of Ang II immunoreactivity, suggesting a role of Ang II in the regulation of gene transcription.
Key Words: angiotensin II • cerebellum • microscopy, electron • gold colloid • cell nucleus
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Introduction |
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Besides its classic function as a cardiovascular hormone, angiotensin II (Ang II) has been discovered to serve as a neurotransmitter and neurohormone.1 2 3 4 Ang II has been localized in areas of the central nervous system known to be important in cardiovascular control,1 2 5 and specific high-affinity peptide receptors have been described mediating the various effects of Ang II on neurons6 and astroglia.7 Most of these actions, ranging from physiological to cellular and genomic responses, are initiated by membrane receptor activation, followed by intracellular signal transduction pathways that are still not fully understood.6 7 A rapid and transient induction of c-fos mRNA expression is, for example, observed after Ang II stimulation of cultured astroglial cells.8
Furthermore, evidence has accumulated that polypeptide ligands and their membrane receptors may have an important additional signaling role within the cell, for example, at the nuclear level (for review see Reference 9). In peripheral tissues, intracellular and nuclear effects of Ang II have been described with the use of various approaches. In isolated hepatocytes, Ang II was found to stabilize angiotensinogen mRNA10 and to stimulate mRNA synthesis specifically,11 12 involving binding to putative nuclear Ang II receptors13 14 and changes in chromatin structure.15 Stimulation of gene transcription was further described for vascular smooth muscle16 17 and endothelial18 cells, suggesting a novel mechanism whereby Ang II could locally and directly influence the permeability, growth, and function of the vascular endothelium independent of changes in hemodynamics.17
Only a few reports exist on direct intracellular localizations of Ang II. Radioactively labeled Ang II was found in nuclei of smooth and cardiac muscle cells19 and vacuole-like structures in different blood cells.20 With the use of Ang II–colloidal gold conjugates, internalization of the peptide into rat vascular smooth muscle cells was studied.21 22 It was convincingly shown that Ang II is taken up into cells by receptor-mediated endocytosis and remains detectable in large lysosome-like vesicles deep within the cell, occasionally in perinuclear regions.
Despite the discovery of a cytosolic angiotensin binding protein23 not representing internalized receptors, the intracellular pathways and cellular functions of Ang II are far from clear. Reports do not agree on the cellular localization of Ang II,24 and information on subcellular structures is often missing. Therefore, in the present study, we aimed to clarify the subcellular localization of the peptide using modern methods of cryoultramicrotomy according to Tokuyasu25 26 with enhanced preservation of structure and antigenicity. Except for the work of Kettani and coworkers,27 who detected renin, angiotensinogen, and Ang II in cytoplasmic granules of pituitary cells, this technique has not been applied to the renin-angiotensin system. In the present study, we chose the rat cerebellar cortex because earlier work28 revealed, especially in this region, remarkable discrepancies in the location of Ang II immunoreactivity with the use of two different primary antibodies. Moreover, the cerebellum seemed well-suited for the application of cryopreparations on neuronal tissue because of its relatively homogeneous organization.
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Animals and Tissue Preparation
Fifteen specific pathogen-free adult male Sprague-Dawley rats (average body weight, 300 g) were used in this study. They were kept under conditions of regular light (lights on at 6 AM, off at 8 PM) and constant temperature (23°C) and had free access to food pellets and tap water.
The rats were deeply anesthetized with sodium pentobarbital (60 mg/kg body wt) and perfused through the left cardiac ventricle with 100 mL of 0.9% saline and 350 mL of fixative, both ice-cold. The following mixtures were used for fixation: (1) 4% formaldehyde (always freshly prepared from paraformaldehyde)/0.2% picric acid in 0.1 mol/L phosphate buffer, pH 6.9 (see Reference 28); (2) 4% formaldehyde in 0.1 mol/L phosphate buffer, pH 7.2, and 0.18 mol/L sucrose; and (3) 4% formaldehyde/0.1% to 2% glutaraldehyde (EM grade, Roth) in 0.1 mol/L phosphate buffer, pH 7.2, and 0.18 mol/L sucrose.
Fixation times were between 5 and 10 minutes. Brains were then quickly removed from the skull, postfixed in the same fixative used for perfusion, and processed for either conventional cryostat sectioning or cryoultramicrotomy. For conventional methods, the whole cerebellum was postfixed in fixative 1 (formaldehyde/picric acid) for 1 hour and kept in 10% sucrose in 0.1 mol/L phosphate-buffered saline (PBS) for 24 to 72 hours at 4°C. The cerebellum then was frozen with CO2, sectioned in a cryostat, and processed for immunohistochemistry as described28 with the use of the avidin/biotin/peroxidase technique.
For cryoultramicrotomy, the region of the crus 1 and 2 ansiform and the paramedian lobules of the cerebellum29 was cut off, dissected into small pieces (approximately 1 mm3), and postfixed for 2 hours at 4°C in fixative 2 or 3 (formaldehyde/glutaraldehyde). After several washes in phosphate buffer and 0.18 mol/L sucrose, the specimens were immersed in 2.3 mol/L sucrose for 24 hours, followed by freezing in liquid nitrogen.
Semithin (1-µm) and ultrathin (40-nm) cryosections were cut with an ultramicrotome (Reichert-Jung Ultracut E) attached to a cryosystem (FC4E) according to Tokuyasu25 26 at -80°C and -110°C, respectively. Usually, semithin cryosections were stained with toluidine blue for orientation in the tissue, followed by ultrathin sectioning of the same block in a selected region.
For light microscopy, semithin sections were placed on gamma-aminopropyltriethoxysilane–activated glass slides. Before immunohistochemical labeling, the remaining sucrose drops were diluted in PBS for 2x10 minutes. Ultrathin cryosections were mounted on Formvar-carbon–coated copper grids.
Antibodies
The monoclonal antibody against Ang II (KAA8) was a gift from Dr T.M. Reilly (DuPont Merck Pharmaceutical Co). KAA8 is a monoclonal, affinity chromatography–purified IgG with an enhanced affinity for Ang II. As demonstrated by Reilly et al30 and Wong et al,31 KAA8 has no cross-reactivity with the angiotensin peptides Ang-(6-8), Ang-(5-8), and Ang-(4-8); 2% cross-reactivity with Ang-(3-8); 32% with Ang-(2-8); and 7% with Ang I [Ang-(1-10)].
The polyclonal antibody against Ang II (K19) was a gift from Dr J. Peters (University of Heidelberg [Germany]). The K19 rabbit anti–Ang II antiserum was obtained by immunization with Ang II coupled to porcine 
-globulin by the carbodiimide method. It has 0.02% cross-reactivity with Ang-(1-10) in the displacement assay and 0.5% cross-reactivity with Ang-(1-10) in the direct binding assay.32
The polyclonal antibody against glial fibrillary acidic protein (GFAP) was obtained from Dakopatts.
Immunocytochemical Labeling
Antibodies were diluted in washing buffer (PBS containing 0.12% glycine and 1% bovine serum albumin [BSA, fraction V; Serva]) to 1:1000 (polyclonal anti–Ang II, K19) or 1:200 (GFAP) or to a final concentration of 5.6 µg/mL (monoclonal anti–Ang II, KAA8). Because of the dense labeling obtained with the monoclonal anti–Ang II, the washing buffer was in some cases supplemented with additional background-reducing agents such as 1% BSA-C (AurIon) instead of normal BSA, with NaCl to a final concentration of 350 mmol/L, or with 0.1% Tween 20.
After the cryosections had been conditioned with washing buffer for 3x5 minutes, primary antibodies were incubated in a humidified chamber for 1 hour at room temperature, followed by five rinses in washing buffer for 3 minutes each. For signal detection, gold-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were used: 12 nm colloidal gold–AffiniPure goat anti-mouse IgG, EM grade, in the case of the monoclonal anti–Ang II; and 18 nm colloidal gold–AffiniPure goat anti-rabbit, EM grade, in the case of the polyclonal anti–Ang II and anti-GFAP. The conjugates were diluted in washing buffer according to the manufacturer's instructions and applied to the sections in a humidified chamber for 30 minutes, followed by five rinses in washing buffer for 3 minutes each.
After removal of the washing buffer in double-distilled water for 4x1 minute, the ultrathin cryosections were contrasted and stabilized with a mixture of 0.3% uranyl acetate and 2% methylcellulose (25 cps; Sigma Chemical Co).
Gold conjugates on semithin cryosections were postfixed with 2% glutaraldehyde in PBS for 15 minutes, followed by intensification with silver acetate according to Hacker33 with the addition of gum arabic (AurIon) to a final concentration of 11%.
Controls
Nonspecific staining was assessed by omission or preadsorption of the primary antibody. In the case of preadsorption, the peptide (human Ang II, Peninsula Laboratories) was added in 100-fold excess (560 µg/mL) to the antibody for 2 hours at room temperature or overnight at 4°C.
Microscopy
Light microscopy was performed with an Axioplan Universal Microscope (Zeiss) equipped with Nomarski differential interference contrast. Electron micrographs were taken with a Philips EM 400T and Jeol 1200 EX at acceleration voltages of 80 and 60 kV, respectively.
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The approach of combining semithin and ultrathin cryosections from one specimen for detection of antigens at the light and electron microscopic levels34 was successfully applied to brain tissue. With these methods, the relatively homogeneous cerebellar cortex could be easily sectioned, and the different layers could be identified even at the subcellular level without any embedding steps known to influence structure and antigenicity.
Conventional fixation with formaldehyde/picric acid or with formaldehyde alone was not applicable to the production of ultrathin cryosections from cerebellar cortical tissue because sections partly disintegrated during spreading. The addition of even small amounts of glutaraldehyde, however, caused a reproducible quality of the sections. Moreover, increasing concentrations of glutaraldehyde led to remarkable changes in Ang II immunoreactivity. If not otherwise indicated, the following results refer to specimens fixed with 4% formaldehyde/0.5% glutaraldehyde.
With formaldehyde/picric acid as a fixative and conventional immunohistochemical techniques such as cryostat sections and enzymatic signal detection, Ang II immunoreactivity was detected as patchy yellow-brownish staining in different layers of the cerebellar cortex, especially the granular layer (Fig 1A
, monoclonal antibody KAA8). The addition of glutaraldehyde to the fixative revealed a nuclear localization of Ang II mainly in Purkinje cells (Fig 1B, arrowheads), whereas the structural preservation in cryostat sections was still not sufficient to resolve the weaker Ang II staining in other cell types (Fig 1B). Only the introduction of semithin cryosections with remarkably better structural preservation enabled the clear localization of Ang II immunoreactivity in the nuclei of Purkinje, granule, basket, and stellate cells (Fig 1C). Because of the use of the highly sensitive particular gold markers, the immunoreactivity could be related to subcellular structures such as the nucleus, with the nucleolus being free of label (Fig 1C, inset). Preadsorption of monoclonal anti–Ang II (KAA8) with the peptide did not yield any signal (Fig 1D), nor did omission of the primary antibody (not shown). Addition of background-reducing agents throughout the incubation did not change the labeling in the case of 0.1% Tween 20 but slightly reduced the immunostaining in the case of the highly stringent, acetylated BSA-C and the high salt concentration. In all cases, the distribution of gold markers over nuclei, vesicles, etc, remained unchanged. It should be emphasized again that Fig 1A through 1D were all made with the same monoclonal anti–Ang II; hence, changes in immunoreactivity were solely due to changes in fixation and structural preservation.
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The preferential localization of Ang II immunoreactivity in neurons (Fig 1C
) can be clearly seen from the distribution of glial cells, stained for GFAP in Fig 1E. In the case of GFAP immunoreactivity, the black signal was located over astrocytes in the granule and Purkinje cell layers (Fig 1E, arrow) and in Bergmann glial processes oriented in parallel perpendicular to the molecular layer (Fig 1E, arrowheads).
At the subcellular level, staining for Ang II existed in nuclei of granule cells, as shown with monoclonal (Fig 2B) and polyclonal (Fig 2C) anti–Ang II. Interestingly, a remarkable dense labeling of the peptide was found at cell-cell contacts in the abutting neuropil (Fig 2B, arrowheads). Corresponding to the results found at the light microscopic level (Fig 1C), the highest intensity of Ang II immunoreactivity was detected in Purkinje cell nuclei, mainly consisting of transcriptionally active euchromatin (Fig 3A). Endothelial cells, occurring numerously throughout the entire cerebellar cortical regions examined, always showed a preferred localization of Ang II immunoreactivity in the euchromatin (Fig 3C), clearly separated from the condensed, darkly contrasted heterochromatin. In contrast to the strong nuclear labeling, Ang II immunoreactivity was occasionally found in the cytoplasm of different cell types, in vesicle-like structures of the cytoplasm (Fig 3A), and in mitochondria (Figs 3C and 4B). Preadsorption of the primary antibody abolished the Ang II immunolabeling in nuclei and cytoplasm of all cell types (Fig 3B). In mitochondria, however, approximately one third of the labeling remained unchanged even after preadsorption of the primary antibody (not shown).
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In addition to the cerebellum, we included in the study additional brain regions (eg, other parts of the cerebellar cortex and median eminence; not shown) and other organs known to have their own renin-angiotensin systems. In all cases, the characteristic nuclear Ang II immunoreactivity could be confirmed, as shown also for rat hepatocytes and cells in the zona glomerulosa of the adrenal gland (Fig 4A and 4B).
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Discussion |
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Immunohistochemical detection of small molecular weight antigens is based on the assumption that fixation immobilizes the substance studied by inducing a coupling reaction with tissue proteins.35 Especially in tissues such as brain, in which not all compartments are directly accessible via the bloodstream, the fixation may be incomplete, so considerable redistribution of the antigen can be expected during fixation, particularly for soluble proteins.36 As far as the octapeptide Ang II is concerned, the subcellular distribution was determined in only one case after fixation with high concentrations of glutaraldehyde.19 Injection of 14C-labeled Ang II into the left cardiac ventricle and subsequent perfusion with 1% glutaraldehyde revealed a rapid and strong autoradiographic signal in nuclei of smooth and cardiac muscle cells.
Immunohistochemical localizations of components of the brain renin-angiotensin system mainly have been made with reversible cross-linking agents such as formaldehyde/picric acid1 2 24 28 37 or with very small amounts of glutaraldehyde added to the fixative.3 27 38 Larsson39 recommended the use of a mixture of 2% formaldehyde and 3% glutaraldehyde for immunocytochemistry of peptides, and a recent study40 demonstrated that the immunoreactivity of several peptides and proteins in brain tissue can be adequately maintained after fixation with 3.5% glutaraldehyde. The present report is the first to use high concentrations (0.5% to 2%) of the irreversibly cross-linking agent glutaraldehyde to retain Ang II in brain tissue. The peptide, most likely cross-linked to tissue proteins via its N-terminus,24 35 was clearly detected in nuclei of neurons after changes in fixation conditions (see Fig 1A and 1B). In the present study, both antibodies used yielded the same result. Previous studies with the same monoclonal anti–Ang II in combination with formaldehyde fixation revealed a putative staining of glial cell populations.28 Ang II immunoreactivity was detected in fibers and cell bodies with the use of polyclonal anti–Ang II with or without affinity purification1 2 24 but no nuclear signals. With formaldehyde/picric acid for fixation, at least a partial redistribution of the antigen cannot be ruled out in these previous studies during the process of sucrose infiltration before cryostat sectioning.
Besides the fixation, the enhanced structural preservation due to cryoultramicrotomy and the use of distinct particular gold markers definitely contributed to the remarkable changes in Ang II immunoreactivity (Fig 1A and 1C). Postfixation of small tissue pieces, shorter infiltration times with sucrose, and the well-preserved antigenicity following the techniques of Tokuyasu25 26 should be mentioned in this context. Among the few reports in this field comparing immunohistochemical methods in detail, the studies of Campbell and coworkers41 revealed that angiotensinogen immunoreactivity in the rat brain strongly depends on the methods of tissue treatment and sectioning.
Localization of Ang II in neurons has been shown for a variety of brain regions,1 2 3 24 37 42 43 but there is also evidence for its occurrence in glial cells.28 However, the present results indicate the existence of Ang II immunoreactivity in neurons such as Purkinje, granule, basket, and stellate cells, clearly separated from the GFAP-stained astrocytes and Bergmann glial processes (Fig 1C and 1E). It remains to be determined whether Ang II is produced within neurons and/or is taken up from the astroglia44 or from the circulating blood. The latter is supported by findings of remarkable amounts of Ang II immunoreactivity in endothelial cells (Fig 3C). The intense Ang II labeling of cell-cell contacts (Fig 2B, arrowheads) points to a transport of the peptide from cell to cell, even through the neuropil. In general, neuropeptides may act as transcellular signals, being released from one neuron and operating within another, provided that the cells have the necessary uptake systems in the plasma and nuclear membranes.45
The rat cerebellum contains all components of the renin-angiotensin system.28 In addition to our findings of strong neuronal Ang II immunoreactivity, especially in Purkinje cells (Fig 1C, inset), renin,46 angiotensin-converting enzyme,47 and the angiotensin-cleaving endopeptidase 24.1548 have been detected in these cells. Hence, an intraneuronal generation and turnover of the peptide seems possible, at least in Purkinje cells, from which Ang II can be either secreted and/or taken up into the nucleus to regulate gene transcription.
Ang II internalization into vascular smooth and heart muscle and endothelial and blood cells has been shown with different high-resolution techniques,19 20 21 22 exhibiting an uptake into lysosomes or smaller vesicle-like structures. In accordance with this, we found Ang II immunoreactivity in the cytoplasm, in vesicle-like structures (Fig 3A), and in mitochondria (Figs 3C and 4B) of brain, liver, and adrenal tissue. The involvement of an intracellular renin-angiotensin system in mitochondrial steroidogenesis has been discussed49 50 with an immunocytochemical localization of renin in intramitochondrial dense bodies. It should be stressed, however, that the mitochondrial labeling found in our studies could not be abolished after preadsorption of the primary antibody with the peptide and therefore may contain nonspecific binding. Further studies are necessary to clarify this point.
Although the exact intracellular pathways are still unclear, the transport of the peptide across the endothelial "barrier" seems to occur very rapidly.19 As has been suggested for insulin and other peptides,9 the Ang II internalization into cells may be followed by specific nuclear binding and accumulation within nuclei.13 A binding to chromatin with putative changes in chromatin conformation and structure15 and a role of the free peptide as a transcellular signal acting as its own second messenger13 are supported by the present localization of Ang II in euchromatin of nuclei (see the figures). This unique nuclear staining observed in neurons and endothelial cells as well as in cells of the liver and adrenal gland should encourage further functional studies on the role of Ang II in the modulation of gene transcription, especially in neuronal networks after its predominant formation in astroglial networks (see Reference 28) and its diffusion in the extracellular fluid involving the communication mode of volume transmission to reach the neurons.44
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Acknowledgments |
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This work was supported by a grant from the Deutsche Akademie der Naturforscher Leopoldina and the Bundesministerium fur Forschung und Technologie to Dr B. Erdmann. We thank Dr Romuald Wroblewski, Karolinska Hospital, and Prof Tomas Hokfelt, Karolinska Institute, Stockholm, for providing the facilities for electron microscopy. We are grateful to Dr Thomas Reilly, DuPont Merck Pharmaceutical Co, Wilmington, Del, for providing monoclonal anti–Ang II (KAA8) and to Dr Jorg Peters, University of Heidelberg, for providing polyclonal anti–Ang II (K19).
Received May 29, 1996; first decision June 27, 1996; accepted June 27, 1996.
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