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Endocrinology Vol. 140, No. 1 500-509
Copyright © 1999 by The Endocrine Society

ARTICLES

Angiotensin II Type 2 Receptor-Mediated Apoptosis of Cultured Neurons from Newborn Rat Brain1

Ujjwala V. Shenoy, Elaine M. Richards, Xian-Cheng Huang and Colin Sumners

Department of Physiology, College of Medicine and the University of Florida Brain Institute, University of Florida, Gainesville, Florida 32610

Address all correspondence and requests for reprints to: Dr. Colin Sumners, Department of Physiology, P.O. Box 100274, 1600 S.W. Archer Road, University of Florida, Gainesville, Florida 32610. E-mail: csumners{at}phys.med.ufl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin II (Ang II) type 2 (AT2) receptors are highly expressed in neonate brain and may have a role in developmental processes such as apoptosis. Concurrent activation of c-Jun N-terminal kinase (JNK) and inhibition of Erk mitogen-activated protein kinase activities is important for apoptosis in many cells, and we previously demonstrated that stimulation of AT2 receptors causes decreased mitogen-activated protein kinase activity in neurons cultured from newborn rat hypothalamus and brain stem. Using such cultures we have employed terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling and internucleosomal DNA fragmentation to assess the role of AT2 receptors in neuronal apoptosis. Ang II (100 nM; 4–72 h) alone produced no significant neuronal apoptosis, and AT2 receptor activation did not stimulate JNK activity. However, exposure of cultures to UV radiation (6 J/m2/sec for 4 sec) to stimulate JNK elicited neuronal apoptosis that was significantly enhanced by Ang II, an effect that was abolished by the AT2 receptor antagonist PD 123,319 (1 µM) or the serine/threonine phosphatase inhibitor okadaic acid (3 nM). Additionally, Ang II enhanced the UV radiation-induced decrease in the levels of the DNA repair enzyme poly-(ADP-ribose) polymerase. These data indicate that Ang II, via AT2 receptors and activation of a serine/threonine phosphatase, contributes to neuronal apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MAMMALIAN tissues contain both angiotensin II (Ang II) type 1 (AT1) and Ang II type 2 (AT2) receptors (1, 2). Most of the well known actions of Ang II on cardiovascular regulation, fluid balance, and hormone secretion are mediated by the AT1 receptors (3, 4, 5). By contrast, the physiological roles of the AT2 receptors remain poorly defined, although the high level of expression of these sites in neonate tissues has led to the suggestion that they may be involved in differentiation and development (6, 7, 8, 9). A number of in vitro and in vivo studies provide support for this idea. For example, it has been demonstrated that stimulation of AT2 receptors by Ang II causes neurite outgrowth in NG108–15 neuroblastoma x glioma and PC-12W pheochromocytoma cells (10, 11) and is antiproliferative in a number of peripheral cell types (12, 13). More recent studies have demonstrated that AT2 receptors may contribute to Schwann cell-mediated myelination and the neuroregenerative responses of dorsal root ganglia that occur after sciatic nerve transection in adult rats (14). Another interesting aspect of AT2 receptors is their transient high expression in fetal rat brain (8, 9), where cell death by apoptosis (programmed cell death) is a prominent feature (15). This high expression of AT2 receptors in various brain regions declines rapidly after birth, suggesting a role for these sites in neuronal apoptosis during central nervous system development. Recent studies have shown that Ang II, via AT2 receptors, induces the death of PC12-W pheochromocytoma cells and human endothelial cells via apoptosis (16, 17), a process that is characterized by cell shrinkage, nuclear condensation, and DNA fragmentation (18). In the case of PC12-W cells, which are used as an in vitro model of sympathetic neurons, the intracellular mechanisms of AT2 receptor-mediated apoptosis include inhibition of Erk mitogen-activated protein (MAP) kinases via induction of the protein tyrosine phosphatase (PTPase) MAP kinase phosphatase 1 (MKP-1) (16). In many cells, including pheochromocytoma cells, inhibition of MAP kinases with simultaneous activation of c-Jun N-terminal kinase (JNK) have been shown to be key events in the induction of apoptosis (19).

To evaluate the signal transduction pathways that are coupled to AT2 receptors, our laboratory has used primary cultured neurons prepared from newborn rat hypothalamus and brain stem. These cultured neurons contain both AT1 and AT2 receptors that are mostly present on different populations of neurons (20, 21, 22) (Sumners, C., and M. Zhu, unpublished data). A limited population of neurons express both AT1 and AT2 receptors (22). Previous studies from our laboratory have demonstrated that selective stimulation of AT2 receptors in these cultured neurons causes an inhibition of Erk1 and Erk2 MAP kinase activities via activation of serine/threonine phosphatase type 2A (PP-2A) (23). Based upon these findings and the known role of MAP kinases in apoptosis, in the present study we determined whether stimulation of AT2 receptors on cultured neurons induced apoptosis. The present data indicate that incubation of cultured neurons with Ang II alone does not induce apoptosis. Further, stimulation of AT2 receptors by Ang II did not cause any change in the activity of JNK. By contrast, in a situation where JNK is strongly stimulated (exposure to UV radiation), Ang II elicited an AT2 receptor-mediated enhancement of neuronal apoptosis, as evidenced by terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL), internucleosomal DNA fragmentation, and decreased levels of the intact form of poly-(ADP-ribose) polymerase (PARP). Furthermore, the data indicate that the effects of Ang II were mediated by a serine/threonine phosphatase, probably PP-2A. These data provide the first indication that Ang II, via AT2 receptors, contributes to the apoptosis of neurons derived from the neonatal rat central nervous system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Newborn Sprague-Dawley rats were obtained from our breeding colony, which originated from Charles River Laboratories, Inc. (Wilmington, MA). DMEM was obtained from Life Technologies (Gaithersburg, MD). Plasma-derived horse serum was obtained from Central Biomedia (Irwin, MO). Losartan potassium (Los) was provided by Merck (Rahway, NJ). PD 123,319 was obtained from Research Biochemicals International (Natick, MA). Ang II, [Sar1,Ile8]Ang II (Sarile), sodium orthovanadate (Van), and normal goat serum were purchased from Sigma Chemical Co. (St. Louis, MO). Okadaic acid (OA) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). CGP 42112A was purchased from Bachem (Torrance, CA). In Situ Cell Death Detection kits, Fast Red tablets, and terminal transferase kits were purchased from Boehringer Mannheim (Indianapolis, IN). 3'-[{alpha}-32-P]Cordyceptin 5'-triphosphate (5000 Ci/mmol) and Renaissance Enhanced Chemiluminescence Detection kits were purchased from DuPont-New England Nuclear (Boston, MA). Protein mol wt markers, 10% polyacrylamide gels, and polyvinylidene difluoride membranes were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Monoclonal anti-PARP antibody (C2–10) and purified intact (116 kDa) and fragmented (89 kDa) PARP from HeLa cells were purchased from Dr. G. G. Poirier (Laval University, Quebec, Canada). Peroxidase-linked rabbit antimouse IgG and Texas Red-linked goat antirabbit antibody were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Polyclonal anti-AT2 receptor antibody was a gift from Dr. S. J. Fluharty (University of Pennsylvania, Philadelphia, PA). DNA labeling dye Hoechst 33258 was obtained from Molecular Probes, Inc. (Eugene, OR). All other chemicals were purchased from Fisher Scientific International, Inc. (Pittsburgh, PA).

Preparation of cultured neurons
Primary cultured neurons were prepared from the hypothalamus and brain stem regions of newborn Sprague-Dawley rats as detailed previously (20). Cells dissociated from the hypothalamus and brain stem were pooled, resuspended in DMEM containing 10% plasma-derived horse serum, plated in 35-mm dishes (3 x 106 cells/dish), and used after 10–15 days in culture. During this period, these cocultures of hypothalamus and brain stem consisted of about 90% neurons and about 10% astroglia and microglia, as estimated by immunocytochemical staining with antibodies against neurofilament proteins and glial fibrillary acidic protein (21). The neurons within these cultures contained specific Ang II receptors, and of these, approximately 70% were AT2 receptors, whereas approximately 30% were AT1 receptors (20). The glia within these neuronal cultures did not contain detectable levels of AT1 or AT2 receptors (20) (Sumners, C., unpublished data).

UV radiation treatment protocol
Cultured neurons were exposed to UV radiation using a Black Ray Lamp (UV Products, Upland, CA) as follows. Growth media were removed from each culture dish and transferred to separate sterile tubes. Each dish was exposed to either high (12 J/m2/sec) or low (6 J/m2/sec) intensity UV light for a period of 4 sec, followed by replacement of the original medium. Control dishes were sham irradiated under identical conditions, i.e. removal and replacement of growth medium.

Detection of apoptosis in cultured neurons
Apoptosis of cultured neurons was assessed by measuring cellular DNA fragmentation using two different methods as follows.

In situ detection of DNA fragmentation by TUNEL.The detection of apoptotic neurons after various treatments was assessed using the TUNEL method, which is based on in situ detection of fragmented DNA, a characteristic of apoptosis (24). After control or experimental treatments, growth media were removed, and the cultured neurons were rinsed once with PBS (pH 7.2). Next, cultures were fixed using freshly prepared 4% paraformaldehyde in phosphate buffer (pH 7.4). In situ detection of apoptotic neurons was performed using an In Situ Cell Death Detection Kit. Briefly, paraformaldehyde-fixed neurons were permeabilized for 4 min on ice with a buffer containing 0.1% Triton X-100 and 0.1% sodium citrate. The permeabilized neurons were then incubated at 37 C for 1 h with TUNEL reaction mixture (terminal deoxynucleotidyl transferase with a fluorescein-tagged substrate). The incorporation of fluorescein was detected by an antifluorescein antibody conjugated with alkaline phosphatase (converter-AP solution). After the substrate reaction (Fast Red substrate), stained neurons (pink color) were analyzed under a light microscope. The number of apoptotic neurons was quantified by counting TUNEL-positive neurons on 10–20 randomly chosen fields (size of each field = 0.09 mm2)/dish in each treatment situation in a blinded fashion. All stained neurons, from those colored light pink, which may be an early apoptotic cell, to dark pink, which may represent a cell in the late stages of apoptosis or an apoptotic body, have been included in our counting protocol without any classification or gradation of intensities. It should be noted that when counting the apoptotic cells, we focused on the cells that had the appearance of neurons based upon our previous morphological and immunostaining experiments (25). In addition, as the neurons are located on top of the glia within the culture dish, we were able to focus the microscope away from the glia and so include only neurons in our counting. The number of apoptotic neurons from each area were combined, and an average value was obtained for each treatment group (generally from duplicate samples). To calculate the total number of neurons per field, we used a Nikon Eclipse E-400 microscope (Nikon, Melville, NY) hooked up to a Sony color video camera (model DXC-970 MD, Sony, Tokyo, Japan). For each culture dish, 5–10 random fields were viewed under the microscope, and each captured image was analyzed using a computer program (microcomputer imaging device program, MCID-M4–3.0, Imaging Research, Inc., Ontario, Canada) to count the total number of neurons in each field. The pooled number of apoptotic neurons from 10–20 different areas in each dish was expressed per 10,000 total neurons. The values from different experimental groups were compared to analyze the effects of specific treatments on apoptosis. The total number of cells in each dish was analyzed across various treatments within each experiment as well as across various experiments to detect dish to dish variation as well as variation within different batches of cells.

Autoradiographic analysis of internucleosomal DNA fragmentation (DNA laddering).The detection of DNA fragmentation was based on the method described by Tilly and Hsueh (26). DNA was isolated after control or experimental treatments and quantitated by UV spectrophotometry. DNA (500 ng) was labeled with 3'-[{alpha}-32P]cordyceptin 5'-triphosphate at the 3'-end of DNA fragments using a terminal transferase enzyme (Boehringer Mannheim, Indianapolis, IN). The labeled DNA was electrophoresed on 2% agarose gels, and then the gels were dried using a slab gel dryer (Fisher Biotech, Pittsburgh, PA). The dried gels were wrapped in plastic wrap and exposed to Kodak X-Omat film (Eastman Kodak Co., Rochester, NY) to detect the 32P-labeled DNA fragments. The extent of apoptosis was qualitatively estimated by comparing the band intensities of the fragmented DNA in each treatment group.

Immunofluorescent localization of AT2 receptors on apoptotic neurons
Immunofluorescent procedures were used to assess whether the apoptotic neurons express AT2 receptors. Cultured neurons were exposed to low intensity UV light followed by incubation with 100 nM Ang II for 48 h in a 37 C incubator. After these incubations, cultures were rinsed with PBS (pH 7.2) and then fixed with freshly prepared 4% paraformaldehyde in phosphate buffer (pH 7.4). Fixed cultures were washed four times (5 min/wash) with PBS containing 0.2% Triton X-100 (PBST), followed by blocking with a buffer containing 5% normal goat serum and 1% BSA in PBST for 2–3 h at room temperature. Cultures were further blocked for 18 h at 4 C with 5% nonfat dry milk in PBST. The localization of AT2 receptors was analyzed using a primary rabbit polyclonal antibody (1:200 dilution, 24 h at 4 C), followed by a secondary Texas Red-tagged antirabbit goat IgG (1:50 dilution, 90 min at room temperature). After the incubation with the secondary antibody, cultures were washed three times with normal saline (5 min/wash) in preparation for the detection of apoptosis. Cultures were then incubated with the DNA-labeling dye Hoechst 33258 (1 µg/ml) for 20 min at room temperature to detect apoptotic neurons. Nonspecific binding of the primary and secondary antibodies was assessed by incubating the cultures with primary antibody alone, secondary antibody alone, or preimmune rabbit serum (protein concentration matched with the dilution of primary antibody) in combination with the secondary antibody. Cultures stained with Hoechst 33258 alone or for AT2 receptors alone were used as additional controls to analyze the fluorescence emitted by Texas Red under the filter used to detect the Hoechst dye staining and the fluorescence emitted by the Hoechst dye under the filter used to detect the Texas red staining. Immunofluorescent neurons were observed using a Zeiss Axioscop microscope (Zeiss, New York, NY).

Analysis of PARP levels in cultured neurons
Levels of intact PARP in cultured neurons were analyzed by Western blots using a specific anti-PARP antibody. Growth media were removed from cultured neurons after control or experimental treatments, and cells were rinsed once with ice-cold PBS (pH 7.2) and were then processed for Western blot analysis as detailed previously (27), using the anti-PARP antibody at a dilution of 1:10,000. Intact and fragmented PARP from HeLa cell extracts were used as positive controls. The band intensities of the intact PARP (113 kDa) were compared across various treatment groups.

Analysis of JNK activity
JNK activity was measured by the ability of extracts from control and experimentally treated cultured neurons to phosphorylate a glutathione-S-transferase (GST)-c-Jun fusion protein, containing amino acids 1–93 of c-Jun with the Ser63 and Ser73 JNK phosphorylation sites. All procedures, including construction of the fusion protein, preparation of cell extracts, and solid phase JNK assay were detailed previously (28).

Drug applications
Ang II and all drugs were first dissolved in the appropriate solvents and were then diluted with PBS, followed by final dilution into the DMEM surrounding the cells. Solvent controls were performed for each experimental protocol. In appropriate treatment groups, UV- or sham-irradiated cultured neurons were incubated with PD 123,319, Los, Sarile, OA, or Van for 10 min at 37 C before treatment with Ang II or PBS.

Statistical analysis
All results are expressed as the mean ± SEM and were obtained by combining data from individual experiments. Multiple means were compared by using one- or two-way ANOVA followed by Newman-Keuls test to assess statistical significance. P < 0.05 was considered significant. Statistical analyses were performed using Sigma Chemical Co. Stat Software (Jandel Scientific, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of Ang II and UV radiation on neuronal apoptosis and JNK activity
In the first series of experiments the effect of Ang II alone on neuronal apoptosis was evaluated by counting TUNEL-positive neurons. Incubation of cultured neurons with Ang II (100 nM) for 4–72 h resulted in a minor increase in apoptosis compared with that in controls (Fig. 1Go, A and B) that was not statistically significant (Fig. 2AGo). The lack of effect of Ang II alone to stimulate apoptosis may be due to its inability to activate one or more of the intracellular pathways that are involved in the process of cell death. It is well known that simultaneous stimulation of JNK and inhibition of Erk1 and Erk2 MAP kinases are critical for the induction of apoptosis in a variety of cell types (19). Previous studies from our laboratory have demonstrated that stimulation of AT2 receptors elicits an inhibition of MAP kinase activities (23). Here we have shown that incubation of cultured neurons for up to 60 min with CGP 42112A, which is a selective AT2 receptor agonist at this concentration (29), caused no significant change in JNK activity as measured by the ability of neuronal extracts to phosphorylate GST-c-Jun (1–93) (Fig. 2BGo). Thus, it is possible that the lack of effect of Ang II alone on apoptosis is due to the fact that it does not activate JNK via AT2 receptors. Based on this, we investigated the effects of Ang II on neuronal apoptosis under conditions where JNK is preactivated. Exposure of neurons to UV radiation, a known stimulator of JNK (30), caused a robust increase in neuronal JNK activity (Fig. 2BGo).



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  		Figure 1. Representative phase contrast micrographs of apoptotic cultured neurons. Phase contrast micrographs were taken from cultured neurons that had been incubated in DMEM for 48 h at 37 C under the following experimental conditions: control solution (PBS; A), 100 nM Ang II (B), and exposure to UV radiation at 6 J/m2/sec for 4 sec followed by PBS (C), 100 nM Ang II (D), 1 µM PD 123,319 plus 100 nM Ang II (E), or 1 µM Sarile plus 100 nM Ang II (F). After these incubations, cultures were prepared for TUNEL staining as detailed in Materials and Methods. Examples of apoptotic neurons (pink) are indicated by the arrows. Bar = 50 µm.

 


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  		Figure 2. Effects of Ang II and UV radiation alone on apoptosis and JNK activity in cultured neurons. A, Duplicate dishes of cultured neurons were incubated in DMEM for 48 h at 37 C under the following experimental conditions: control solution (PBS; Con), 100 nM Ang II (Ang II), exposure to UV radiation at 6 J/m2/sec for 4 sec followed by PBS (low UV), and exposure to UV radiation at 12 J/m2/sec for 4 sec followed by PBS (high UV). Cultured neurons then underwent TUNEL staining as detailed in Materials and Methods. TUNEL-positive neurons were counted on 20 randomly chosen areas (size of each area = 0.09 mm2)/dish in each treatment situation. All counting was performed via blinded evaluation of the dishes. The number of apoptotic neurons from each area were combined and are plotted on the y-axis as the number of apoptotic neurons per 10,000 cells. Data are the mean ± SEM from three different experiments. *, P < 0.05 compared with controls. B, Quadruplicate dishes of cultured neurons were incubated for the indicated times in DMEM at 37 C in the absence (Con) or presence of 10 nM CGP42112A or were exposed to UV radiation as a positive control (UV; 6 J/m2/sec for 4 sec) followed by incubation in DMEM for 30 min. Incubations were followed by analysis of JNK activity as detailed in Materials and Methods. Top, A representative autoradiogram showing bands corresponding to 32P-labeled GST-c-Jun-(1–93) in each treatment situation (JNK phosphorylates this fusion protein at Ser63 and Ser73, and so the extent of phosphorylation indicates the extent of JNK activity). Bottom, Bar graph showing the quantification of the 32P-labeled GST-c-Jun-(1–93) bands (except UV). Data are the mean ± SEM from 4 different experiments and are presented as percentages of the control levels (defined as 100%).

 
To evaluate the possibility that Ang II may cause apoptosis in cells in which JNK is activated, we used UV radiation as a stimulator of JNK activity. A series of preliminary studies were carried out to evaluate an exposure time and intensity of UV radiation that would result in a significant level of apoptosis with a minimal level of necrosis. Cultured neurons were exposed to low (6 J/m2/sec) or high (12 J/m2/sec) intensity UV radiation for 4 sec. The extent of apoptosis was evaluated 48 h later by counting TUNEL-positive neurons in each treatment situation. Exposure of cultured neurons to low intensity UV radiation alone resulted in a significant increase in the number of apoptotic (TUNEL-positive) neurons present after 48 h compared with that in controls without any significant level of necrosis (Figs. 1CGo and 2AGo). Similarly, exposure of cultures to high intensity UV radiation alone resulted in a significant increase in the number of apoptotic (TUNEL-positive) neurons present after 48 h compared with that in control cultures (Figs. 1CGo and 2AGo). However, there was a significant level of necrosis in the cultures exposed to high intensity UV radiation, resulting in detachment of cells from culture dishes. Thus, exposure of cultures to low intensity UV radiation was used in subsequent experiments. In preliminary experiments we determined that coincubation of UV-irradiated cultures for 48 h with 1, 10, and 100 nM Ang II resulted in concentration-dependent enhancement of UV radiation-induced neuronal apoptosis (data not shown). The highest and statistically significant effect was observed with 100 nM Ang II; hence, in subsequent experiments we used 100 nM Ang II. Exposure of cultures to UV radiation for 8, 24, or 48 h produced a time-dependent increase in neuronal apoptosis (Fig. 3Go). A significant enhancement of UV radiation-induced neuronal apoptosis was obtained after a 48-h incubation with Ang II, but at shorter incubation times the effect was not statistically significant (Figs. 1DGo and 3Go). These results indicate that Ang II alone has no significant effect on apoptosis of cultured neurons, but causes a significant enhancement of UV radiation-induced neuronal apoptosis. Based upon this, the above treatment conditions (low intensity UV radiation for 4 sec followed by 100 nM Ang II) and an incubation time of 48 h were used to induce apoptosis in all subsequent experiments.



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  		Figure 3. Ang II enhances the stimulatory effects of UV radiation on apoptosis of cultured neurons. Effects of Ang II and UV radiation as a function of incubation time. Duplicate dishes of cultured neurons were incubated in DMEM for 8, 24, or 48 h at 37 C under the following experimental conditions: control solution (PBS; Con) and exposure to UV radiation at 6 J/m2/sec for 4 sec followed by either PBS (UV) or 100 nM Ang II (UV/AngII). After the above incubations, cultured neurons underwent TUNEL staining as detailed in Materials and Methods. TUNEL-positive neurons were counted on 10 randomly chosen areas (size of each area = 0.09 mm2)/dish in each treatment situation, and all counting was performed via blinded evaluation of the dishes. The number of apoptotic neurons from each area were combined and are plotted on the y-axis as the number of apoptotic neurons per 10,000 cells. Data are the mean ± SEM from 3 different experiments. *, P < 0.05 compared with controls. 2+, P < 0.05 compared with UV alone. **, P < 0.05 compared with UV, 8 and 24 h.

 
Ang II-induced enhancement of neuronal apoptosis is mediated via AT2 receptors
In the next set of experiments we evaluated the role of specific Ang II receptors in the Ang II-induced enhancement of neuronal apoptosis. Preincubation of cultures with the nonselective Ang II receptor antagonist Sarile (1 µM) for 10 min completely abolished the enhancement of UV radiation-induced neuronal apoptosis by Ang II (100 nM), as evidenced by TUNEL-positive staining (Figs. 1FGo and 4AGo). Similar inhibitory effects were obtained by pretreating the cultures with the selective AT2 receptor blocker PD 123,319 (1 µM) for 10 min (Figs. 1EGo and 4AGo). In addition, in cultures pretreated with the selective AT1 receptor blocker Los (1 µM), Ang II (100 nM) elicited a significant enhancement of UV radiation-induced neuronal apoptosis that was completely abolished by 1 µM PD 123,319 (Fig. 4BGo). Neither PD 123,319 (Fig. 4BGo) nor Sarile (not shown) alone had a significant effect on neuronal apoptosis. The role of AT2 receptors in apoptosis of cultured neurons was confirmed in experiments using internucleosomal DNA fragmentation as an index of neuronal programmed cell death (18). In these experiments sham- or UV-irradiated cultured neurons were pretreated with Los (1 µM) for 10 min, followed by exposure to PBS or Ang II (100 nM) for 48 h. No apparent DNA fragmentation was observed in the control neurons (Fig. 5Go), in agreement with the TUNEL staining data. In the UV-radiated neurons, fragments of DNA in multiples of about 185 bp (the size of a nucleosome) were observed, and coincubation of UV-radiated neurons with Ang II produced an enhancement of this effect, as demonstrated by the increased intensity of the bands corresponding to the 185-bp DNA fragments (Fig. 5Go). Collectively, these data indicate that the Ang II-induced enhancement of UV radiation-stimulated apoptosis is mediated via AT2 receptors.



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  		Figure 4. Ang II enhancement of UV radiation-induced neuronal apoptosis is mediated via AT2 receptors. A, Effects of Sarile and PD 123,319 on Ang II-induced neuronal apoptosis. Duplicate dishes of cultured neurons were incubated in DMEM for 48 h at 37 C under the following experimental conditions: control (PBS; Con); exposure to UV radiation at 6 J/m2/sec for 4 sec followed by PBS (UV), 100 nM Ang II (UV/AngII), 1 µM PD 123,319 plus 100 nM Ang II (UV/PD/Ang II), 1 µM PD 123,319 (UV/PD), 1 µM Sarile plus 100 nM Ang II (UV/Sar/Ang II), or 1 µM Sarile (UV/Sar). B, Effects of PD 123,319 on Ang II-induced neuronal apoptosis in the presence of Los. Duplicate dishes of cultured neurons were incubated in DMEM for 48 h at 37 C under the following experimental conditions: control (PBS; Con) and exposure to UV radiation at 6 J/m2/sec for 4 sec followed by PBS (UV), 100 nM Ang II (UV/AngII), 1 µM PD 123,319 plus 100 nM Ang II (UV/PD/Ang II), 1 µM PD 123,319 (UV/PD), or 1 µM PD 123,319 after sham radiation (PD). All incubations were performed in the presence of Los (1 µM). After the above incubations in A and B, cultured neurons underwent TUNEL staining as detailed in Materials and Methods. TUNEL-positive neurons were counted on 10 randomly chosen areas (size of each area = 0.09 mm2)/dish in each treatment situation, and all counting was performed via blinded evaluation of the dishes. The number of apoptotic neurons from each area were combined and are plotted on the y-axis as the number of apoptotic neurons per 10,000 cells. Data are the mean ± SEM from 3 different experiments. *, P < 0.05 compared with controls; 2+, P < 0.05 compared with UV alone.

 


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  		Figure 5. Ang II enhances UV radiation-induced internucleosomal DNA fragmentation via AT2 receptors. Cultured neurons were incubated in DMEM for 48 h at 37 C in the presence of 1 µM Los under the following experimental conditions: control (PBS; lane 1) and exposure to UV radiation 6 J/m2/sec for 4 sec followed by either PBS (UV; lane 2) or 100 nM Ang II (UV/Ang II; lane 3). After these incubations, DNA was isolated from the cultures and was nick end labeled with 3'-[a32P]cordyceptin-5'-triphosphate as detailed in Materials and Methods. The autoradiogram shown here is representative of three experiments.

 
Apoptotic neurons contain AT2 receptors
Coimmunostaining of UV radiation/Ang II (100 nM)-treated cultures revealed that 85–90% of the apoptotic neurons present exhibited AT2 receptor immunofluorescence. Figure 6AGo is a phase contrast micrograph of a field of cultured neurons, identified by morphological analysis and previous immunostaining experiments with antineurofilament antibodies (25). Figure 6BGo is an immunofluorescence micrograph of the same field of neurons stained with the DNA-labeling dye Hoechst 33258. The arrows in Fig. 6BGo indicate apoptotic neurons, as evidenced by the presence of condensed apoptotic nuclei. Figure 6CGo is an immunofluorescence micrograph of the same field of neurons stained with the AT2 receptor antibody. It can be seen that the apoptotic neurons in Fig. 6BGo exhibit AT2 receptor immunofluorescence. A small number of apoptotic neurons did not exhibit AT2 receptor immunofluorescence (data not shown). It should also be noted that none of the control conditions/incubations noted in Materials and Methods produced any specific immunofluorescence.



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  		Figure 6. Localization of AT2 receptors on apoptotic neurons. Cultured neurons were incubated in DMEM for 48 h at 37 C after exposure to UV radiation 6 J/m2/sec for 4 sec followed by 100 nM Ang II (UV/Ang II). Cultures were then prepared for immunofluorescent staining as detailed inMaterials and Methods. A is a phase contrast micrograph of a field of cultured neurons, identified by morphological analysis and previous immunostaining experiments with antineurofilament antibodies (25 ). B is an immunofluorescence micrograph of the same field of neurons stained with the DNA labeling dye Hoechst 33258. The arrows indicate apoptotic neurons, as evidenced by the presence of condensed nuclear matter. C is an immunofluorescence micrograph of the same field of neurons stained with the AT2 receptor antibody. The arrows indicate that the apoptotic neurons seen in B are positive for AT2 receptor immunofluorescence. Bar = 20 µm.

 
Ang II enhances the UV radiation-induced reduction in levels of PARP in cultured neurons
Cleavage of the DNA repair enzyme PARP into inactive fragments by cysteine-aspartate proteases (caspases) such as CPP-32 is known to contribute to neuronal apoptosis (31). In this set of experiments we determined whether treatment with UV radiation and Ang II elicited any change in the levels of intact PARP protein in cultured neurons. Cultures were incubated with control solution or were exposed to UV radiation in the absence or presence of 100 nM Ang II for 4, 24, or 48 h. In the rat, intact PARP is a 113-kDa protein, and Western immunoblots using the C2–10 anti-PARP antibody revealed bands of this size, compared with the 116-kDa band observed in HeLa cells (Fig. 7Go). Under the experimental conditions used here, the C2–10 antibody did not recognize the 89-kDa cleavage fragment of PARP in the cultured neurons, even though this antibody has been used to detect fragmented PARP in other studies (32, 33). Four hours after exposure of cultured neurons to UV radiation in the absence or presence of Ang II, there were no changes in the levels of intact PARP compared with those in control neurons (data not shown). However, 24 and 48 h after exposure of cultured neurons to UV radiation alone, there was a clear decrease in the levels of intact PARP compared with those in control neurons (Fig. 7Go). The reduction in the levels of intact PARP produced by UV radiation was enhanced at both time points by coincubation with 100 nM Ang II (Fig. 7Go). Ang II alone produced no changes in the levels of intact PARP (data not shown). These data provide further evidence that the DNA fragmentation detected with TUNEL staining after UV radiation and Ang II treatments is due to apoptosis rather than transient DNA damage.



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  		Figure 7. Ang II enhances the UV radiation-induced reduction in the levels of intact PARP in cultured neurons. Cultured neurons were incubated in DMEM at 37 C for 24 or 48 h under the following experimental conditions: control (PBS; Con) and exposure to UV radiation at 6 J/m2/sec for 4 sec followed by either PBS (UV) or 100 nM Ang II (UV/AngII). After these incubations, total proteins were isolated from the cultures and were subjected to gel electrophoresis (40 µg/lane) alongside intact PARP from HeLa cells (~25,000 cells/lane). Changes in the levels of intact PARP in cultured neurons in each treatment situation were analyzed by Western blot analysis as detailed in Materials and Methods. Shown here is a Western immunoblot that is representative of 3 different experiments. Lane 1, 116-kDa intact PARP from HeLa cells. Lanes 2–7, 113-kDa intact PARP from neurons under the following conditions: lane 2, Con, 24 h; lane 3, UV, 24 h; lane 4, UV/Ang II, 24 h; lane 5, Con, 48 h; lane 6, UV, 48 h; lane 7, UV/Ang II, 48 h.

 
The enhancement of UV radiation-induced neuronal apoptosis produced by Ang II involves a serine/ threonine phosphatase
Inhibition of MAP kinases is an important factor in the induction of apoptosis in various cell types (16, 19). Previous studies from our laboratory indicate that stimulation of AT2 receptors elicits a decrease in MAP kinase activity in cultured neurons via activation of PP-2A (23). By contrast, in PC-12W and N1E-115 neuroblastoma cells, Ang II elicits an AT2 receptor-mediated stimulation of PTPase activity (16, 34, 35). In fact, induction of the PTPase MKP-1 is responsible for the inhibition of MAP kinase activity observed in PC-12W cells in response to Ang II (16). For these reasons we evaluated the possible roles of both serine/threonine phosphatase and PTPases in the enhancement of UV radiation-induced neuronal apoptosis by Ang II. UV- or sham-irradiated cultured neurons were preincubated with either control solution or the serine/threonine phosphatase inhibitor OA (3 nM) for 15 min before incubation with Ang II (100 nM) or control solution. At this concentration OA is a selective inhibitor of PP-2A (36). TUNEL staining revealed that 3 nM OA did not elicit any significant neuronal apoptosis by itself under these conditions and did not alter the level of neuronal apoptosis in cultured neurons exposed to UV radiation alone (Fig. 8Go). However, OA completely reversed the enhancement of UV radiation-induced neuronal apoptosis produced by Ang II (Fig. 8Go). Preincubation of UV- or sham-irradiated cultured neurons with the PTPase inhibitor Van (10 mM) for 10 min before treatment with Ang II did not alter the enhancement of UV radiation-induced apoptosis produced by this peptide (Fig. 8Go). TUNEL staining also revealed that Van alone did not elicit significant neuronal apoptosis (Fig. 8Go). Collectively, these results suggest that the enhancement of UV radiation-induced neuronal apoptosis by Ang II involves activation of a serine/threonine phosphatase, but not a PTPase.



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  		Figure 8. Ang II enhancement of UV radiation-induced neuronal apoptosis is mediated via activation of a serine/threonine phosphatase. Effects of OA and Van on Ang II-induced neuronal apoptosis. Duplicate dishes of cultured neurons were incubated in DMEM at 37 C for 48 h under the following experimental conditions: control (PBS; Con); exposure to UV radiation at 6 J/m2/sec for 4 sec followed by PBS (UV), 100 nM Ang II (UV/AngII), 3 nM OA plus 100 nM Ang II (UV/OA/AngII), 3 nM OA (UV/OA), 10 µM Van plus 100 nM Ang II (UV/Van/AngII), or 10 µM Van (UV/Van); and sham exposure to UV radiation followed by 3 nM OA or 10 µM Van. After these incubations cultured neurons underwent TUNEL staining as detailed inMaterials and Methods. TUNEL-positive neurons were counted on 10 randomly chosen areas (size of each area = 0.09 mm2)/dish in each treatment situation, and all counting was performed via blinded evaluation of the dishes. The number of apoptotic neurons from each area were combined and are plotted on the y-axis as the number of apoptotic neurons per 10,000 cells. Data are the mean ± SEM from three different experiments. *, P < 0.05 compared with controls; 2+, P < 0.05 compared with UV alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented here indicate that Ang II alone does not cause significant apoptosis of neurons cultured from newborn rat hypothalamus and brain stem. However, Ang II does elicit a significant enhancement of the neuronal apoptosis that is caused by exposure of these cultures to UV radiation, as evidenced by an increased number of apoptotic neurons and internucleosomal DNA fragmentation. Furthermore, this stimulatory effect of Ang II is mediated via AT2, not AT1, receptors. Our data also indicate that a great majority of the apoptotic neurons contain AT2 receptors, suggesting that the effects of Ang II are directly on the neuron that is undergoing apoptosis. The presence of a small population of apoptotic neurons that do not contain AT2 receptors might also mean that in some instances the effects of Ang II are indirect, i.e. Ang II acting at its AT2 receptors may release a paracrine substance that acts at a neuron nearby to elicit apoptosis. An alternative explanation for the presence of a population of apoptotic neurons that do not contain AT2 receptors is that these are cells that are undergoing apoptosis due to exposure to UV radiation without the added influence of Ang II.

The intracellular mechanisms that mediate the above stimulatory actions of Ang II on neuronal apoptosis include the activation of a serine/threonine phosphatase. This conclusion is based upon the fact that the actions of Ang II on neuronal apoptosis are abolished by OA. Based upon our previous demonstration that Ang II elicits an AT2 receptor-mediated stimulation of PP-2A in these neurons (27) and that the concentration of OA used here is quite selective for PP-2A (36), we would speculate that this particular serine/threonine phosphatase is involved in the enhancement of UV radiation-induced neuronal apoptosis produced by Ang II. The inability of Van to block the enhancement of UV radiation-induced neuronal apoptosis by Ang II indicates that tyrosine phosphatases are not involved in this response. This is in contrast to the situation in PC-12W cells, where Ang II elicits apoptosis via a mechanism that includes induction of a tyrosine phosphatase, MKP-1, and inhibition of MAP kinases (37). The findings from PC-12W cells are certainly consistent with the idea that in certain cell types an inhibition in the activity of MAP kinases is an important event in the induction of apoptosis (19). It is well established that either MKP-1 or PP-2A can inhibit Erk MAP kinases (38, 39), and our previous studies indicate that the AT2 receptor-mediated stimulation of PP-2A in cultured neurons elicits an inhibition of Erk1 and Erk2 (23). Thus, the involvement of PP-2A in the Ang II-induced enhancement of UV radiation-induced neuronal apoptosis may reside in its ability to inhibit MAP kinases (37). It is apparent that in certain cells, a simultaneous stimulation of JNK activity along with the inhibition in MAP kinase activity is crucial for apoptosis (19). Considering that activation of neuronal AT2 receptors does not alter JNK activity (Fig. 2BGo), it is not surprising that Ang II alone did not elicit apoptosis. This may explain why a stimulatory effect of Ang II on neuronal apoptosis was only observed in a situation where JNK is activated, e.g. exposure of cultures to UV radiation (Fig. 2BGo) (30). The exposure of cultured neurons to UV radiation in the present studies is clearly an artificial situation, and the question remains as to the identity of physiological or pathological agents that stimulate JNK and along with Ang II ultimately elicit apoptosis. It has been suggested that the initial signaling event that causes activation of the JNK cascade in response to UV radiation involves the multimerization and clustering of cell surface receptors for growth factors and cytokines (40). Further, UV radiation induces the expression of proinflammatory cytokines such as interleukin-1 and tumor necrosis factor (40), and administration of these cytokines to HeLa cells causes a robust activation of JNK (40). Thus, specific cytokines or growth factors may work in concert with Ang II and, by virtue of their respective effects on JNK and MAP kinases, elicit apoptosis. The nature of these physiological or pathological activators of JNK will be the subject of our future studies.

Increases in JNK activity lead to the phosphorylation and activation of the tumor suppressor protein p53 (41), which, in turn, has been demonstrated to elicit apoptosis in many cell types, including neurons (42). The activation of p53 causes apoptosis via modulation of a cascade of enzymatic processes that ultimately result in an imbalance between proapoptotic and antiapoptotic proteins. Included among these proteins are the caspases, which play a primary role in neuronal apoptosis (43). For example, studies suggest that activation of a member of the caspase family, CPP32, plays a role in the apoptosis of granule neurons, but not in their death due to necrosis (31). This activation of CPP32 leads to apoptosis via the hydrolysis and inactivation of PARP (31), a nuclear protein that is involved in the DNA repair process and whose breakdown via proteolysis is associated with apoptosis (44). In the present study, exposure of cultured neurons to UV radiation resulted in a decrease in the levels of intact PARP protein, and this effect was enhanced by coincubation of neurons with Ang II. The decrease in the levels of intact PARP was observed 24 h before the detection of DNA fragmentation by TUNEL staining. This suggests that the DNA fragmentation observed in these studies may involve inactivation of PARP, possibly due to its metabolism via proteolysis. However, as the PARP fragments could not be detected under our experimental conditions, it might also be argued that the decrease in the intact form of PARP is due to a reduction in the synthesis of this protein. In any case, a net decrease in the intact form of PARP and hence a net decrease in its activity suggest that the DNA fragmentation observed with TUNEL staining is due to apoptosis rather than to transient DNA damage. It is interesting to note that the AT2 receptor-mediated apoptosis of endothelial cells involves activation of CPP32 (17). Although it is possible that the decrease in the levels of intact PARP in cultured neurons is mediated via the activation of CPP32 by Ang II, this will only be confirmed by further experiments. Although we were not able to detect any changes in PARP protein levels 4 h after UV radiation/Ang II treatment, an increase in PARP protein/activity earlier than 4 h and the resulting energy depletion of energy substrates such as NAD cannot be completely ruled out. Such energy depletion may contribute to cell death via apoptosis (45).

In summary, our data indicate a role for Ang II, acting via AT2 receptors, in neuronal apoptosis. In addition, our data suggest a role for PP-2A and PARP in this effect of Ang II, although the exact cellular mechanisms involved remain to be evaluated. As AT2 receptors are expressed at high levels in neonatal brain, our data further support the idea that these receptors may be involved in apoptosis associated with neuronal development and differentiation (1, 6, 9).


   Acknowledgments
 
The authors thank Jennifer Moore for preparation of cultured neurons, Shirley Johnson for typing the manuscript, and Dr. Steven J. Fluharty for generously providing the AT2 receptor antibody.


   Footnotes
 
1 This work was supported by a grant from the NIH (NS-19441) and a postdoctoral fellowship (to U.V.S.) from the American Heart Association, Florida Affiliate. Back

Received April 1, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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