Light-induced Photoreceptor Apoptosis in Vivo Requires Neuronal Nitric-oxide Synthase and Guanylate Cyclase Activity and Is Caspase-3-independent
2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês
10.1074/jbc.m005359200
ISSN1083-351X
AutoresMaryanne Donovan, Ruaidhrı́ J. Carmody, Thomas G. Cotter,
Tópico(s)bioluminescence and chemiluminescence research
ResumoApoptosis is the mode of photoreceptor cell death in inherited and induced retinal degeneration. However, the molecular mechanisms of photoreceptor cell death in human cases and animal models of retinal dystrophies remain undefined. Exposure of Balb/c mice to excessive levels of white light results in photoreceptor apoptosis. This study delineates the molecular events occurring during and subsequent to the induction of retinal degeneration by exposure to white light in Balb/c mice. We demonstrate an early increase in intracellular calcium levels during photoreceptor apoptosis, an event that is accompanied by significant superoxide generation and mitochondrial membrane depolarization. Furthermore, we show that inhibition of neuronal nitric-oxide synthase (nNOS) by 7-nitroindazole is sufficient to prevent retinal degeneration implicating a key role for neuronal nitric oxide (NO) in this model. We demonstrate that inhibition of guanylate cyclase, a downstream effector of NO, also prevents photoreceptor apoptosis demonstrating that guanylate cyclase too plays an essential role in this model. Finally, our results demonstrate that caspase-3, frequently considered to be one of the key executioners of apoptosis, is not activated during retinal degeneration. In summary, the data presented here demonstrate that light-induced photoreceptor apoptosis in vivo is mediated by the activation of nNOS and guanylate cyclase and is caspase-3-independent. Apoptosis is the mode of photoreceptor cell death in inherited and induced retinal degeneration. However, the molecular mechanisms of photoreceptor cell death in human cases and animal models of retinal dystrophies remain undefined. Exposure of Balb/c mice to excessive levels of white light results in photoreceptor apoptosis. This study delineates the molecular events occurring during and subsequent to the induction of retinal degeneration by exposure to white light in Balb/c mice. We demonstrate an early increase in intracellular calcium levels during photoreceptor apoptosis, an event that is accompanied by significant superoxide generation and mitochondrial membrane depolarization. Furthermore, we show that inhibition of neuronal nitric-oxide synthase (nNOS) by 7-nitroindazole is sufficient to prevent retinal degeneration implicating a key role for neuronal nitric oxide (NO) in this model. We demonstrate that inhibition of guanylate cyclase, a downstream effector of NO, also prevents photoreceptor apoptosis demonstrating that guanylate cyclase too plays an essential role in this model. Finally, our results demonstrate that caspase-3, frequently considered to be one of the key executioners of apoptosis, is not activated during retinal degeneration. In summary, the data presented here demonstrate that light-induced photoreceptor apoptosis in vivo is mediated by the activation of nNOS and guanylate cyclase and is caspase-3-independent. retinitis pigmentosa nitric oxide nitric-oxide synthase N G-nitro-l-arginine methyl ester N G-nitro-d-arginine methyl ester phosphate-buffered saline 7-nitroindazole 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one terminal dUTP nick end labeling cyclic guanosine monophosphate terminal deoxynucleotidyl transferase hydroethidine 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolecarbocyanine iodide reactive oxygen species mitochondrial membrane potential outer nuclear layer inner nuclear layer Acetyl-Asp-Glu-Val-Asp-p-nitroanilide Asp-Glu-Val-Asp-fluoromethylketone caspase-activated DNase poly(ADP-ribose)polymerase fetal calf serum 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid permeabilization buffer polyacrylamide gel electrophoresis Retinitis pigmentosa (RP)1, refers to a group of hereditary disorders of the retina characterized by a progressive loss of rod and cone photoreceptors. Nyctalopia (night-blindness), an initial symptom of the disease is associated with rod photoreceptor degeneration. As the disease progresses, severe loss of peripheral and central fields occur, due to the subsequent loss of cone photoreceptors. The genetics of RP is complex and to date at least 30 genes have been implicated in the etiology of RP, the majority of which encode photoreceptor-specific proteins that are either components of the phototransduction cascade or structural components of these cells (see the Retinal Information Network available on the University of Texas School of Public Health Web server). Despite the diverse genetics underlying the pathology of RP, one feature common to both human cases and animal models is the apoptotic cell death of rod and cone photoreceptors (1Chang G.Q. Hao Y. Wong F. Neuron. 1993; 11: 595-605Abstract Full Text PDF PubMed Scopus (567) Google Scholar, 2Li Y. Chopp M. Jiang N. Yao F. Zaloga C. J. Cereb. Blood Flow Metab. 1995; 15: 389-397Crossref PubMed Scopus (464) Google Scholar). Apoptosis, therefore, represents a common final pathway in the pathology of RP. Exposure to excessive levels of white light induces photoreceptor apoptosis and has previously been used as a model for the study of retinal degeneration (3Reme C.E. Weller M. Szczesny P. Munz K. Hafezi F. Reinboth J. Clausen M. Anderson R.E. Degenerative Diseases of the Retina. Plenum Press, New York1995: 19-25Crossref Google Scholar). Furthermore, several animal studies have demonstrated that photoreceptors from retinal degeneration mutants are more susceptible to the damaging effects of excessive light than normal photoreceptors (4Naash M.L. Peachey N.S. Li Z.Y. Gryczan C.C. Goto Y. Blanks J. Milam A.H. Ripps H. Invest. Ophthalmol. Vis. Sci. 1996; 37: 775-782PubMed Google Scholar, 5LaVail M.M. Gorrin G.M. Yasumura D. Matthes M.T. Invest. Ophthalmol. Vis. Sci. 1999; 40: 1020-1024PubMed Google Scholar, 6Sanyal S. Hawkins R.K. Vision Res. 1986; 26: 1177-1185Crossref PubMed Scopus (61) Google Scholar, 7Chen J. Simon M.I. Matthes M.T. Yasumura D. LaVail M.M. Invest. Ophthalmol. Vis. Sci. 1999; 40: 2978-2982PubMed Google Scholar). The evidence from these animal models suggests that excessive light may enhance the progression and severity of some forms of human RP. The mechanism by which light induces retinal degeneration is at present unclear. A recent study demonstrated that rhodopsin is essential for light-induced retinal degeneration, indicating that signal flow through the phototransduction cascade is necessary to mediate the damaging effects of light (8Grimm C. Wenzel A. Hafezi F., Yu, S. Redmond T.M. Reme C.E. Nat. Genet. 2000; 25: 63-66Crossref PubMed Scopus (227) Google Scholar). Indeed, constitutive signal flow in phototransduction is thought to underlie some forms of inherited retinal disorders (9Fain G.L. Lisman J.E. Exp. Eye Res. 1993; 57: 335-340Crossref PubMed Scopus (90) Google Scholar). This is supported by the significant number of mutated genes that encode components of the phototransduction cascade and are implicated in the etiology of RP or of related conditions. These include rhodopsin, cyclic GMP phosphodiesterase, the rod cGMP-gated channel, and rhodopsin kinase (see the Retinal Information Network available on the Web). Although the exact mechanism of photoreceptor apoptosis resulting from exposure to excessive levels of light remains undefined, a diverse range of agents have been used to prevent photoreceptor apoptosis in this model (10Li Z.Y. Tso M.O. Wang H.M. Organisciak D.T. Invest. Ophthalmol. Vis. Sci. 1985; 26: 1589-1598PubMed Google Scholar, 11Lambiase A. Aloe L. Graefes. Arch. Clin. Exp. Ophthalmol. 1996; 234: S96-S100Crossref PubMed Google Scholar, 12Cayouette M. Behn D. Sendtner M. Lachapelle P. Gravel C. J. Neurosci. 1998; 18: 9282-9293Crossref PubMed Google Scholar). These include calcium channel blockers (13Edward D.P. Lam T.T. Shahinfar S. Li J. Tso M.O. Arch. Ophthalmol. 1991; 109: 554-562Crossref PubMed Scopus (53) Google Scholar) as well as antioxidants (14Lam S. Tso M.O. Gurne D.H. Arch. Ophthalmol. 1990; 108: 1751-1757Crossref PubMed Scopus (58) Google Scholar, 15Rosner M. Lam T.T. Fu J. Tso M.O. Arch. Ophthalmol. 1992; 110: 857-861Crossref PubMed Scopus (27) Google Scholar) implicating a role for both intracellular calcium and reactive oxygen species in retinal apoptosis. However, the molecular basis for the rescue of photoreceptors from apoptosis in light-induced retinal degeneration by these agents has yet to be elucidated. In this study, the molecular events that occur during light-induced photoreceptor apoptosis are delineated. We demonstrate that inhibition of NOS not only prevents photoreceptor cell death, but also additional features of apoptosis, including superoxide generation, mitochondrial membrane depolarization, and elevated intracellular calcium levels, implicating a key role for NO in this model. In addition, the potential involvement of cyclic guanosine monophosphate (cGMP), a central component of the phototransduction cascade, is explored by examining the effects of ODQ (1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one), a potent and selective inhibitor of guanylate cyclase. Inhibition of guanylate cyclase in this study prevents light-induced retinal degeneration. These results suggest that NO mediates cell death in this model through activation of guanylate cyclase, resulting in increased levels of intracellular calcium through cGMP-gated calcium channels. Finally, our results demonstrate that caspase-3 is not activated during photoreceptor apoptosis in this model. Adult male Balb-c mice were maintained in the dark for 18 h before being exposed to constant light. Immediately prior to light exposure their pupils were dilated with 5% cyclopentolate. The mice were then exposed to 2 h of cool white fluorescent light at a luminescence level of 5000 lux. The mice were sacrificed after treatment by cervical dislocation at the following time points: 30 min and 1 h after light onset, immediately after light exposure (0 h) and after 6, 14, and 24 h of darkness that followed the 2-h light exposure. Mice were injected intraperitoneally with the following:N G-nitro-l-arginine methyl ester (l-NAME), 100 mg kg−1 (Sigma Chemical Co., UK) in PBS or the inactive isomerN G-nitro-d-arginine methyl ester (d-NAME), 100 mg kg−1 (Sigma, UK) in PBS as a control, 7-nitroindazole (7NI), 100 mg kg−1 (Sigma, UK) in peanut oil or peanut alone as a control, and ODQ, 50 mg kg−1 (Calbiochem) in Me2SO or Me2SO alone as a control. All intraperitoneal injections were administered 1 h prior to light exposure. Jurkat T-cells were maintained in RPMI containing 10% FCS. 32D cells were cultured in RPMI containing 10% FCS and 10% WEHI-conditioned media. Agents used to induce apoptosis were anti-human Fas (300 ng/ml) (Upstate Biotechnology Inc., Lake Placid, NY) and exposure to ultraviolet (UV) irradiation (10 min). DNA strand breaks in photoreceptor nuclei were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Briefly, enucleated eyes were fixed in 10% buffered formalin for 24 h, dehydrated, processed, and embedded in paraffin. Sections (5 μm) were incubated in 50 μl of reaction buffer containing 2.5 mm CoCl2, 0.1 unit/ml terminal deoxynucleotidyl transferase (TdT) in a 0.1m sodium cacodylate (pH 7.0) buffer and 0.75 nmfluorescein-12-dUTP (Roche Molecular Biochemicals, Germany). These sections were incubated at 37 °C for 1 h in a humidified chamber. Following several washes in PBS, the sections were mounted in mowiol (Calbiochem) and viewed under a fluorescence microscope (Nikon Eclipse E600) using a fluorescein isothiocyanate filter. Three animals were used for each of the time points; 0, 6, 14, and 24 h after light exposure. Enucleated eyes were placed in PBS, and retinal dissection was carried out using a watchmaker's forceps. The choroid, sclera, and pigmented epithelium were removed, and the retina was then separated from the vitreous and lens. Retinal DNA was isolated following phenol-chloroform extraction. Briefly, retinas were placed in 150 μl of lysis buffer (20 mmEDTA, 100 mm Tris, and 0.8% sodium lauryl sarcosinate) containing proteinase K (20 μg/ml) and incubated at 50 °C for 18 h, vortexing occasionally. The DNA was then extracted with phenol chloroform and chloroform isoamyl alcohol (v/v, 24:1). DNA was precipitated with ethanol, and the pellet was dissolved in Tris-EDTA. Total RNase-treated DNA was visualized by including ethidium bromide (0.5 μg/ml) in the agarose and observed by illuminating on a 302-nm UV transilluminator. Retinal dissection was carried out as above. Tissue dissociation was achieved in a 0.25% trypsin solution (Life Technologies, Inc., Paisley, UK). The cells were washed in PBS and fixed in 1% paraformaldehyde at 4 °C for 30 min. The cells were then washed again with PBS and then with permeabilization buffer (PB: 10 mm HEPES, 150 mm NaCl, 4% FCS, 0.1% sodium azide, and 0.1% Triton X-100). Cells were resuspended in PB containing 0.125 μg of anti-active caspase-3 antibody (PharMingen International, San Diego, CA), or the same concentration of an isotype control (rabbit IgG, Sigma, UK) and incubated for 1 h at 4 °C. Following two washes in PB, cells were resuspended in 20 μg/ml fluorescein isothiocyanate-conjugated secondary antibody (goat anti-rabbit, Sigma, UK) and incubated for 1 h at 4 °C. After a further two washes in PB, cells were resuspended in 0.5 ml of PBS for FACScan analysis on a Becton-Dickinson FACScan flow cytometer. Retinas were dissected and washed with cold PBS. Total protein was obtained by homogenizing retinas in 50 μl of chilled lysis buffer containing 10 mm HEPES, pH 7.4, 2 mm MgCl2, 5 mm EGTA, 50 mm NaCl, 1 mmphenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, and 2 μg/ml leupeptin. The cells were incubated on ice for 20 min and then lysed by 3–4 cycles of freezing and thawing. Insoluble material was pelleted by centrifuging at 20,000 × g for 15 min at 4 °C. The protein content of each sample was determined by the Bio-Rad protein assay (Bio-Rad, Hemel Hempstead, UK) using bovine serum albumin as a standard, and 80 μg of protein in 50 μl of lysis buffer was dispensed into each well of a microtiter plate. An equal volume of 2× reaction buffer (50 mm HEPES, pH 7.4, 0.2% CHAPS, 20% glycerol, 2 mm EDTA, and 10 mm dithiothreitol) was added to each sample with 50 μm caspase-3 substrate-DEVD-pNA (Bahcem, Saffron Walden, UK; 1 mm stock in Me2SO). Reactions were incubated at 37 °C for 1 h and then cleavage of the peptide substrate DEVD-pNA was monitored by liberation of the chromogenicpNA in a SpectraMax-340 plate reader (Molecular Devices, CA) by measuring absorption at 405 nm. The retina was dissected, and total protein was obtained by lysing in radioimmune precipitation buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EGTA, 1 mm sodium orthovanadate, 1 mm sodium fluoride) containing antipain (1 μg/ml), aprotinin (1 μg/ml), chymostatin (1 μg/ml), leupeptin (0.1 μg/ml), pepstatin (1 μg/ml), and phenylmethylsulfonyl fluoride (0.1 mm). The amount of total protein of each sample was determined by the Bio-Rad protein assay (Bio-Rad, Hemel Hempstead, UK) using bovine serum albumin as a standard. 60–80 μg of total protein from each sample was electrophoresed on polyacrylamide gels followed by transfer to nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) and incubated overnight with the appropriate antibodies. Antibodies reactive to caspase-3 (Upstate Biotechnology Inc.), ICAD (Oncogene), PARP (PharMingen), nNOS (Transduction Laboratories), iNOS and eNOS (Santa Cruz Biotechnology) were used in this study. Membrane development was achieved using Enhanced Chemiluminescence (ECL) (Amersham Pharmacia Biotech, Buckinghamshire, UK). Superoxide anion levels were measured using a modified version of the assay as previously described (16Gorman A. McGowan A. Cotter T.G. FEBS Lett. 1997; 404: 27-33Crossref PubMed Scopus (206) Google Scholar). Briefly, cells (5 × 105) were loaded with 10 μm hydroethidine (DHE) (Molecular Probes), prepared from a 10 mm stock in Me2SO for 15 min at 37 °C. Superoxide anion oxidizes DHE intracellularly to produce ethidium bromide, which fluoresces upon interaction with DNA. Superoxide anion levels were assessed by monitoring the fluorescence due to ethidium bromide on a Becton-Dickinson FACScan flow cytometer with excitation and emission settings of 488 and 590 nm, respectively. Mitochondrial membrane depolarization was analyzed using the probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolecarbocyanine iodide (JC-1, Molecular Probes). At high mitochondrial membrane potential JC-1 forms J-aggregates, which fluoresce strongly at 590 nm (measured in FL-2). Cells were incubated with JC-1 (5 μg/ml) in darkness for 15 min at 37 °C, and fluorescence was measured on a Becton-Dickinson FACScan flow cytometer with excitation at 488 nm. Intracellular calcium levels were determined using the intracellular calcium probe, Fluo-3 AM (acetoxymethyl ester) (Molecular Probes, Leiden, The Netherlands). Cells were incubated in darkness with Fluo-3 (250 nm), prepared from a 500 μm stock for 15 min at 37 °C, and fluorescence was measured in FL-1 (530 nm) on a Becton-Dickinson FACScan flow cytometer with excitation at 488 nm. Exposure to excessive levels of light results in photoreceptor apoptosis (3Reme C.E. Weller M. Szczesny P. Munz K. Hafezi F. Reinboth J. Clausen M. Anderson R.E. Degenerative Diseases of the Retina. Plenum Press, New York1995: 19-25Crossref Google Scholar). In the present study, Balb/c mice were exposed to 2 h of cool white fluorescent light at a luminescence level of 5000 lux. DNA strand nicking in the outer nuclear layer was assessed by Terminal dUTP nick end labeling (TUNEL) over 24 h in control animals and in animals exposed to 2 h of white fluorescent light. Retinas of control animals showed no TUNEL-positive labeling in photoreceptor nuclei immediately after light exposure (Fig.1 A). At 6 h, positive labeling increased, and at 14 and 24 h, abundant TUNEL-positive cells were apparent in the ONL (Fig. 1, b–e). Apoptotic cell death was confirmed by DNA agarose gel electrophoresis (Fig.1 B). Biochemically, apoptosis is characterized by internucleosomal DNA cleavage-producing DNA fragments that are multiples of 180–200 bp, which appear in agarose gel electrophoresis as a ladder pattern. The presence of this typical ladder pattern at 14 and 24 h (lane 3 and 4) confirms apoptotic cell death in this model and verifies the efficacy of the TUNEL assay to detect apoptosis following light exposure. To elucidate the molecular mechanism of light-induced photoreceptor apoptosis we investigated the role of NOS, previously shown to be involved in another model of retinal degeneration where rats were exposed to light for 7 days at an illuminescence of 90 footcandles (17Goureau O. Jeanny J.C. Becquet F. Hartmann M.P. Courtois Y. Neuroreport. 1993; 5: 233-236Crossref PubMed Scopus (56) Google Scholar). To investigate the role of NOS in this model of light-induced photoreceptor apoptosis, animals were treated with the NOS inhibitor l-NAME (100 mg kg−1), 1 h prior to light exposure. In treated animals no TUNEL-positive photoreceptor cells were detected immediately following light exposure or following a further incubation of 6, 14, and 24 h in darkness (Fig. 2,e–h). Animals that were injected with d-NAME, the inactive isomer of l-NAME, showed similar TUNEL labeling to untreated light-induced animals (Fig. 2, a–d). These results suggest a key role for NO in this model of light-induced retinal degeneration. A role for calcium has previously been implicated in retinal cell death in vivo. The ability of calcium channel blockers to prevent rod photoreceptor apoptosis in two different models of retinal degeneration has been demonstrated (13Edward D.P. Lam T.T. Shahinfar S. Li J. Tso M.O. Arch. Ophthalmol. 1991; 109: 554-562Crossref PubMed Scopus (53) Google Scholar, 18Frasson M. Sahel J.A. Fabre M. Simonutti M. Dreyfus H. Picaud S. Nat. Med. 1999; 5: 1183-1187Crossref PubMed Scopus (203) Google Scholar). However, measurement of calcium levels during photoreceptor apoptosis has not previously been conducted. In this study, intracellular calcium levels were analyzed using the fluorescent probe Fluo-3 AM (Fig. 3). Increased levels of calcium were detected after 30 min of light exposure, and the number of cells with elevated calcium continued to increase up to 3 h. This was followed by a significant decrease in intracellular calcium concentration. These data demonstrate that calcium elevation is an early and rapid event in retinal degeneration. This transient elevation of intracellular calcium is blocked by the NOS inhibitor l-NAME, suggesting that elevated intracellular calcium requires NOS activity. A key role for ROS has been demonstrated in an in vitro model of retinal degeneration, where antioxidants prevent photoreceptor cell death (19Carmody R.J. McGowan A.J. Cotter T.G. Exp. Cell Res. 1999; 248: 520-530Crossref PubMed Scopus (95) Google Scholar).In vivo, a number of compounds possessing antioxidant properties have prevented photoreceptor apoptosis (see "Discussion"). However, to date, little is known about the oxidative pathways involved in such retinal death, nor have ROS measurements been conducted in an in vivo model of retinal degeneration. In this study, superoxide anion formation was monitored using the probe DHE in control light-induced Balb/c mice and in those treated with l-NAME (Fig.4 a). Increased levels of superoxide were detected following 30 min of light exposure, and the number of cells with elevated superoxide levels increased up to 3 h after the light insult. This was followed by a decrease in intracellular levels. This data demonstrates, therefore, that superoxide production is an early event in light-induced retinal degeneration occurring alongside increased calcium levels. Furthermore, our results show that superoxide generation is inhibited byl-NAME, suggesting that NOS activity is also required for the generation of superoxide. Mitochondria are the major site of cellular ROS production. The increase in superoxide production observed here prompted the investigation of mitochondrial dysfunction as alterations in mitochondrial membrane potential (Δψm) can result in increased ROS generation. The lipophilic probe JC-1 was used to analyze Δψm. In the presence of an intact Δψm, JC-1 forms J-aggregates, which are associated with a shift in fluorescence emission (590 nm). Thus, a reduction in fluorescence emission at 590 nm can be interpreted as a reduction in Δψm. As illustrated in Fig. 4 b, a reduction in Δψm is evident in a significant number of cells after 30 min of light exposure and 3 h after light exposure 75% of cells have reduced Δψm. This reduction in Δψm is blocked by l-NAME. Although the NOS inhibitor l-NAME is an established inhibitor of in vivo NO production (20Moore P.K. Oluyomi A.O. Babbedge R.C. Wallace P. Hart S.L. Br. J. Pharmacol. 1991; 102: 198-202Crossref PubMed Scopus (414) Google Scholar),l-NAME shows little or no selectivity for individual NOS isoforms. As both constitutive and inducible isoforms of NOS have been reported in the retina (see "Discussion"), it is unclear which of the three isoforms; inducible nitric-oxide synthase (iNOS), endothelial nitric synthase (eNOS), or neuronal nitric oxide (nNOS) is responsible for the production of NO following light exposure. Indeed, elevated levels of NO may be due to increased NOS protein expression or activation by calcium entry into the cells. Immunoblot analysis revealed no detectable increase in either iNOS or eNOS expression (Fig.5 A, part a) and Fig. 5 A (part b). However, a time-dependent increase in nNOS expression was evident (Fig. 5 A, part c). Densitometric analysis revealed a 2.5-fold increase in nNOS expression at 3 h, a 3-fold increase at 6 h, a 3.5-fold increase at 14 h, and a 5-fold increase at 24 h. The retinas taken from mice treated with 100 mg kg−1 of l-NAME 1 h prior to light exposure showed no increase in nNOS expression (Fig. 5 A,part d). These results suggest that initial NOS activity is required for up-regulation of nNOS. To clarify the role of nNOS in light-induced retinal degeneration, we investigated the effects of treatment with 7NI, a selective inhibitor of neuronal nitric-oxide synthase that has been employed in several in vivo studies to inhibit the activity of neuronal NOS. 7 NI has high selectivity for nNOS (IC50710 nm) over iNOS (IC50 20 μm). In addition, several studies have demonstrated that 7NI had no effect on eNOS in the brain in a number of animal species, including rodents (21Moore P. Wallace P. Gaffen Z. Hart S.L. Babbedge R.C. Br. J. Pharmacol. 1993; 110: 219-224Crossref PubMed Scopus (462) Google Scholar, 22Kelly P.A. Ritchie I.M. Arbuthnott G.W. J. Cereb. Blood Flow Metab. 1995; 15: 766-773Crossref PubMed Scopus (83) Google Scholar, 23Wang Q. Pelligrino D.A. Baughman V.L. Koenig H.M. Albrecht R.F. J. Cereb. Blood Flow Metab. 1995; 15: 774-778Crossref PubMed Scopus (159) Google Scholar). Animals were treated with 100 mg kg−1 of 7NI or peanut oil 1 h prior to light exposure. Retinas of control animals (injected with peanut oil) had scattered TUNEL labeling at 6 h and abundant TUNEL labeling at 14 and 24 h (Fig. 5 B, a–d) similar to untreated light-induced animals (Fig. 1 A). No TUNEL-positive photoreceptor cells were detected at 6, 14, and 24 h in the retina of animals treated with 7NI (Fig. 5 B,e–h). 7 NI, therefore, prevents light-induced photoreceptor apoptosis thus establishing the role of neuronal NOS in promoting light-induced retinal degeneration. A major action of NO is to activate the soluble form of the enzyme guanylate cyclase (24Ignarro L.J. Blood Vessels. 1991; 28: 67-73PubMed Google Scholar, 25Hobbs A.J. Trends Pharmacol. Sci. 1997; 18: 484-491Abstract Full Text PDF PubMed Scopus (244) Google Scholar). Binding of NO to guanylate cyclase increases the latter's activity leading to the formation of cGMP, which may result in excess calcium influx through cGMP-gated channels. To explore the potential involvement of cGMP in photoreceptor apoptosis, we examined the effect of ODQ, a potent and selective inhibitor of NO-sensitive guanylate cyclase. Animals were treated with 50 mg kg−1 of ODQ or Me2SO 1 h prior to light exposure. In the Me2SO control animals, positive TUNEL labeling was apparent at 6, 14, and 24 h (Fig.6, a–d), similar to untreated animals (Fig. 1 A). In animals treated with ODQ, no TUNEL-positive photoreceptor cells were detected at 6, 14, and 24 h (Fig. 6, e–h). These results demonstrate that guanylate cyclase activity is required for light-induced retinal degeneration. Caspase-3 is recognized as one of the key executioners of apoptosis. Recently, activation of caspase-3 was reported during photoreceptor apoptosis in transgenic rats with the rhodopsin mutation S334ter (26Liu C. Li Y. Peng M. Laties A.M. Wen R. J. Neurosci. 1999; 19: 4778-4785Crossref PubMed Google Scholar). In these mutants, pretreatment with the irreversible caspase-3 inhibitor, z-DEVD-fmk prevents photoreceptor apoptosis. However, emerging evidence now suggests that not all cell types require caspase-3 to undergo apoptosis, and it has been reported that oxidative stress can inhibit caspase activation during photoreceptor apoptosis in vitro (27Carmody R.J. Cotter T.G. Cell Death Differ. 2000; 7: 282-291Crossref PubMed Scopus (126) Google Scholar). Because the free radical NO appears to play a key role in light-induced retinal degeneration, we determined the activation status of caspase-3 during photoreceptor apoptosis in this model, using three different analytical techniques (Fig. 7). Caspase-3 is synthesized as a 32-kDa inactive proenzyme and is cleaved at Asp28-Ser29 and Asp175-Ser176 to generate active subunits of 17 and 12 kDa. Analysis of the levels of the 32- and 17-kDa caspase-3 species using Western blot demonstrates the absence of active, 17-kDa caspase-3 in cell lysates taken from the retinas of light-induced Balb/c mice 3, 6, and 14 h after light exposure (Fig.7 A). The murine hematopoietic 32D cell line is included as a control to demonstrate the processing of pro-caspase-3 (32 kDa) to the active 17-kDa fragment as these cells undergo apoptosis following ultraviolet light irradiation. The activity of caspase-3-like proteases was further assessed by measuring the cleavage of the colorimetric substrate AcDEVD-pNA (Fig. 7 B). Again no evidence of AcDEVD-pNA cleavage was obtained in light-induced Balb/c mice 3, 6, or 14 h after light exposure. Untreated and anti-Fas IgM-treated Jurkat cells served as negative and positive controls, respectively. To further confirm the absence of active caspase-3 in the retina of light-induced Balb/c mice, assessment of active caspase-3 content by flow cytometry was carried out using an anti-active caspase-3 antibody (Fig. 7 C). This antibody is raised against the active fragment and preferentially recognizes active caspase-3. Untreated and anti-Fas IgM-treated Jurkat cells served as negative and positive controls, respectively. There was no detectable active caspase-3 at 3, 6, or 14 h after light exposure. Western blot analysis of two caspase-3 substrates, ICAD (inhibitor of caspase-activated Dnase) and PARP (poly (ADP-ribose)polymerase) was carried out to verify the lack of caspase-3 activity during light-induced retinal degeneration. ICAD is a caspase-3 substrate that exists as a complex with CAD and is cleaved at two sites by caspase-3 during apoptosis resulting in the release of CAD. PARP is cleaved and inactivated by caspase-3 into 25- and 85-kDa fragments. Analysis of the levels of the cleaved products of ICAD (12 kDa) and PARP (85 kDa) by Western blot demonstrates the absence of these fragments in cell lysates taken from the retinas of light-induced BALB/C mice 3, 6, and 14 h after light exposure (Fig. 7,D and E). The muri
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