October 2004
Volume 45, Issue 10
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Retinal Cell Biology  |   October 2004
Metipranolol Blunts Nitric Oxide-Induced Lipid Peroxidation and Death of Retinal Photoreceptors: A Comparison with Other Anti-Glaucoma Drugs
Author Affiliations
  • Neville N. Osborne
    From the Nuffield Laboratory of Ophthalmology, Oxford University, Oxford, United Kingdom.
  • John P. M. Wood
    From the Nuffield Laboratory of Ophthalmology, Oxford University, Oxford, United Kingdom.
Investigative Ophthalmology & Visual Science October 2004, Vol.45, 3787-3795. doi:10.1167/iovs.04-0147
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      Neville N. Osborne, John P. M. Wood; Metipranolol Blunts Nitric Oxide-Induced Lipid Peroxidation and Death of Retinal Photoreceptors: A Comparison with Other Anti-Glaucoma Drugs. Invest. Ophthalmol. Vis. Sci. 2004;45(10):3787-3795. doi: 10.1167/iovs.04-0147.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine the effect of the nitric oxide donor sodium nitroprusside (SNP) on rat retinas and to see whether detrimental changes could be attenuated by known antiglaucoma drugs.

methods. SNP was injected into the rat eye and retinas were analyzed by the terminal-deoxynucleotidyl transferase dUTP-linked nick end labeling (TUNEL) procedure and by immunohistochemistry. In some instances, retinal homogenates were analyzed by immunoblot for proteins associated with either photoreceptors or with cell death. Analysis of lipid peroxidation in retinal homogenates was by the thiobarbituric acid reactive species (TBARS) formation method.

results. SNP caused an increase in the number of retinal photoreceptors labeled for DNA breakdown by the TUNEL procedure and for caspase-3 and Bcl-2. After intravitreal injection of SNP, breakdown of poly(ADP-ribose) polymerase and an increase in the level of active forms of caspase-3 and Bcl-2 were detected. Furthermore, photoreceptor-specific rhodopsin kinase was reduced. SNP also stimulated formation of TBARS in retinal homogenates, occurring to a greater extent in retinas from young Royal College of Surgeons rats lacking photoreceptor degeneration. This supports the view that the photoreceptors are the prime target for SNP. Significantly, of several antiglaucoma drugs tested only metipranolol and its active metabolite, desacetylmetipranolol, blunted the SNP-induced retinal changes.

conclusions. Of all antiglaucoma drugs tested, only metipranolol was able to attenuate SNP-induced lipid peroxidation and activation of apoptosis in photoreceptors. Because oxidative injury has been implicated in the pathogenesis of certain ocular diseases, these findings could prove to be of clinical significance.

Nitric oxide (NO) is an inorganic gas that is present in biological systems and has a half-life of a few seconds. At physiological concentrations (described to be in the range of 0.1–100 nM), NO is relatively unreactive and functions as a neuromodulator/neurotransmitter. 1 At higher concentrations, however, NO may be converted to a number of more reactive derivatives, known collectively as reactive nitrogen species, to influence cell death in diverse ways. 2 3 The role of NO in cell death is complex, counteracting the death process in some cases, yet inducing the death process in others. It seems that the outcome is dependent on the local NO concentration, the nature of the target cell, the type of cell injury, and the concomitant presence of other factors. 2 3 This duality in the effect of NO is recognized in the retina where the potential of NO to contribute to or protect against neuron death is clearly documented. 4 5 6 7  
The predominant cell type in the retina is the photoreceptor. These are particularly susceptible to free radical damage or lipid peroxidation, 8 9 10 11 because retinal photoreceptor membranes have an unusually high concentration of docosahexaenoic acid (22:6, ω3) which amounts to nearly 50% of the total fatty acid pool. 12 13 By virtue of its structure, docosahexaenoic acid is especially prone to lipid peroxidation, 14 and for this reason retinal photoreceptors are much more susceptible to NO when compared with other retinal cell-types. 15 For example, Ju et al., 15 injected the antihypotensive agent sodium nitroprusside (SNP) into the aqueous humor of the rat and particularly found the occurrence of photoreceptor death. SNP injected into the aqueous is unlikely to penetrate the vitreous. This strongly supports the view that NO, which is spontaneously generated from SNP, 16 is produced in the aqueous chamber and can diffuse into the vitreous to ultimately cause death of photoreceptors. 15 Photoreceptor death can also be brought about experimentally in albino rats by excessive light, 11 injection of S-antigen to elicit experimental autoimmune uveitis (EAU), 17 or ischemia. 18 19 The cause in all cases appears to be the excessive production of free radicals. That the free radical gas NO is a major player in the death process is implied by the finding that inhibitors of nitric oxide synthase (NOS) attenuate photoreceptor death caused by light 11 or ischemia. 6 7  
Photoreceptors die in age-related macular degeneration (AMD), in which the involvement of free radicals such as NO is therefore strongly suspected. 10 In retinal ischemia (caused by, for example, diabetes, central retinal artery occlusion, glaucoma), any photoreceptor damage is also likely to involve free radicals. Any drug that can attenuate free-radical–induced activation of the apoptotic pathway associated with photoreceptors in experimental studies, especially when the induction is by NO, may therefore be of potential use in the treatment of these diseases. As a prelude to this study, we investigated whether any of the presently used antiglaucoma drugs can act as antioxidants by attenuating SNP and iron/ascorbate-induced lipid peroxidation in brain homogenates. 20 To our surprise, only a single substance, the β-adrenergic receptor antagonist metipranolol, was found to act in this manner. 20 The goal of this study was therefore to extend these studies to the retina with the purpose of elucidating whether metipranolol is the sole antiglaucoma drug able to attenuate SNP/NO-induced photoreceptor apoptosis. We concentrated on the use of SNP for the in vivo studies (as a donor of NO) rather than iron/ascorbate, because NO has been suggested to participate in photoreceptor death in certain situations, as described earlier. 
Methods
Procedures used in this study conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Home Office in the United Kingdom. Rats housed in a 12-hour light–dark cycle were used for all experiments; food and water were provided ad libitum. 
Injection of SNP into the Eye
Adult Wistar rats (200–250 g) were anesthetized by intramuscular injection of a combination of diazepam (0.4 mL/kg) and fentanyl-fluanisone (0.3 mg/kg; Hypnorm; Janssen, Grove, UK). Pupils were dilated by topical application of 1% tropicamide drops. SNP (1 mM) was dissolved in normal saline, and a 5-μL volume injected as a bolus (vitreous humor) or continuously over a period of 15 minutes (aqueous humor) by syringe (Hamilton, Reno, NV). The estimated concentration of SNP injected into the vitreous or aqueous chamber was 50 μM. Control eyes were injected with saline. In some instances SNP was co-injected with 100 μM metipranolol or Trolox (Hoffman-LaRoche, Nutley, NJ) into the vitreous humor. Three days after injection the animals were killed, their retinas fixed in 2% paraformaldehyde, and 10-μm frozen sections cut for immunohistochemistry and staining for breakdown of DNA (using the terminal-deoxynucleotidyl transferase dUTP-linked nick end labeling, or TUNEL, procedure). 
Immunohistochemistry
Frozen retinal sections were thawed and incubated overnight at 4°C with mouse anti-choline acetyltransferase monoclonal (ChAT; 1:100; Sigma-Aldrich, Poole, UK), sheep anti-neuronal nitric oxide synthase (nNOS; 1:2000; gift from Piers Emson, University of Cambridge, UK), mouse monoclonal anti-caspase-3 recognizing the cleaved active form (clone 46; 1:100; BD Biosciences, Cowley, UK), or mouse anti-Bcl-2 monoclonal (1:100; Clone C-2; Santa Cruz Biotechnology Inc., Santa Cruz, CA) antibodies. These were developed with appropriate secondary antibodies conjugated to fluorescein, as previously described. 21  
The TUNEL Procedure for Breakdown of DNA
The method used was essentially as described previously. 22 Briefly, thawed frozen sections were incubated for 5 minutes at room temperature in 30 mM Tris/HCl (pH 7.8). Incubation was continued at 37°C for 10 minutes in the presence of pepsin (800 U/mL) to expose the free DNA ends. After the pepsin was washed away with Tris/HCl, sections were preincubated for 10 minutes with buffer A (30 mM Tris/HCl [pH 7.2], 140 nM sodium cacodylate, and 1 mM cobalt chloride) and then placed in a humid chamber for 60 minutes at 37°C with buffer A containing 0.2 U/μL terminal deoxynucleotidyl transferase (TdT; Roche Diagnostics, Livingston, Scotland, UK) and 15 μM biotin-16-dUTP (Roche Diagnostics). The reaction was stopped by a 15-minute wash in sodium citrate buffer (300 nM NaCl, 30 mM sodium citrate [pH 7.3]) at room temperature. Sections were then rinsed for 10 minutes in Tris-buffered saline (TBS: 20 mM Tris, 150 mM NaCl [pH 7.3]), for a further 10 minutes in TBS solution containing 1% bovine serum albumin, and finally for 10 to 20 minutes with Cy3-conjugated streptavidin. A final washing step was in TBS buffer. Sections were then mounted in buffered glycerol containing phenylenediamine to reduce fluorescence fading. 
Measurement of Lipid Peroxidation in Rat Retinal Extracts and Whole Retinas
Adult Wistar rats or Royal College of Surgeons (RCS) rats were killed by decapitation. Retinas from several Wistar rats were homogenized in 10 volumes of ice-cold 0.9% saline (pH 7.0), by motor-driven polytetrafluoroethylene-glass homogenizer. The homogenate was centrifuged at 1000g for 10 minutes at 4°C, and 0.5 mL of the resultant low-speed supernatant was used for assaying lipid peroxidation. Also, individual retinas from Wistar or RCS rats were placed in 0.5 mL ice-cold 0.9% saline and homogenized by sonification for 2 minutes. To each of the 0.5-mL retinal samples was added 0.3 mL of 0.9% saline (pH 7.0) containing a defined concentration of drug followed by a preincubated period of 5 minutes at 37°C. Lipid peroxidation was then initiated by the addition of SNP (generally 50 or 100 μM) or buffer. The process was terminated after 45 minutes of incubation at 37°C by placing tubes on ice. The degree of lipid peroxidation in each sample was determined by measurement of thiobarbituric acid species (TBARS), as described previously. 23 TBARS are the colored products resulting from the reaction of thiobarbituric acid with compounds formed during lipid peroxidation such as malondialdehyde. Briefly, the color reaction was developed by the sequential addition of 0.2 mL of 8.1% wt/vol sodium dodecyl sulfate, 1.5 mL of 20% vol/vol acetic acid (pH 3.5) and 1.5 mL of 0.8% wt/vol thiobarbituric acid. This mixture was incubated for 30 minutes in a boiling water bath. After cooling with tap water, 2 mL of n-butanol:pyridine (15:1 vol/vol) was added and the reaction mixture centrifuged at 4000g for 10 minutes. Absorbance of the organic layer was measured at a wavelength of 532 nm and the amount of TBARS determined by using a standard curve of the malondialdehyde derivative 1,1,3,3-tetraethoxypropane (1–30 nanomoles). Protein concentration in retinal homogenate supernatant was determined with a bicinchoninic acid protein assay kit (Sigma-Aldrich) with bovine serum albumin used as the standard. 
Electrophoresis and Western Blot Analysis
Retinas were homogenized in freshly prepared 20 mM Tris/HCl buffer (pH 7.4) containing 2 mM EDTA, 0.5 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride. An equal volume of sample buffer (62.5 mM Tris/HCl [pH 7.4], containing 4% SDS, 10% glycerol, 10% β-mercaptoethanol, and 0.002% bromophenol blue) was added, and samples were boiled for approximately 3 minutes. An aliquot was taken for determination of protein content. Electrophoresis of samples by the method of Laemmli 24 was performed on 10% polyacrylamide gels containing 0.1% SDS. Samples were transferred onto nitrocellulose, as previously described. 25 The nitrocellulose blots were incubated with mouse anti-caspase-3 monoclonal (clone 46; 1:100), goat anti-Bcl-2 monoclonal (clone C-2; 1:100), mouse anti-PARP (poly(ADP-ribose) polymerase; recognizing the uncleaved form; 1:400; BD Biosciences), rabbit anti-actin (1:1000; Chemicon, Chandler’s Ford, UK), and rabbit anti-rhodopsin kinase (1:1000; Affinity Bioreagents, Cambridge, UK) for three hours at room temperature, and appropriate secondary antibodies conjugated to horseradish peroxidase were subsequently used. Nitrocellulose blots were developed with a 0.016% solution of 3-amino-9-ethylcarbazole in 50 mM sodium acetate (pH 5) containing 0.05% Tween-20 and 0.03% H2O2
Caspase-3 Assay
Retinal extracts were assayed for caspase-3 activity using a colorimetric activity assay kit (Chemicon) that provides a means for specifically assaying the activity of caspases that recognize the peptide sequence DEVD (caspase-3). 
Nitrite Determination
Lipid peroxidation was induced to retinal homogenates by the addition of SNP (100 μM) or FeCl2 (25 μM) and ascorbate (100 μM) (see Melena and Osborne 20 ) as described already. The homogenates was then analyzed for nitrite content (reflecting NO release) by the Griess reaction. In brief, sample aliquots (50 μL) in 96-well microplates were mixed with 50 μL sulfanilamide (1% wt/vol) in 5% (vol/vol) orthophosphoric acid and incubated for 5 to 10 minutes. N-1-naphthylenediamine dihydrochloride (0.1% wt/vol) was added and absorbance read at 570 nM with a microplate reader (MS212; Titertek Plus, Huntsville, AL). Sodium nitrite was used as a standard to calculate nitrite concentrations. 
Results
Formation of TBARS and Nitrite In Vitro
SNP stimulated TBARS formation in rat retinal homogenates in a dose-dependent manner (Fig. 1a) . The SNP-induced stimulation of TBARS by 100 μM SNP in retinal homogenates was significantly less to that of FeCl2 (25 μM) and ascorbate (100 μM). Moreover, of all the substances tested, only metipranolol, its active metabolite desacetylmetipranolol and Trolox significantly attenuated SNP-induced formation of TBARS (Table 1) . The inhibitory action of metipranolol and desacetylmetipranolol on SNP-induced TBARS formation was concentration dependent (Fig. 1b) with IC50 of 3.6 and 3.8 μM, respectively. SNP also significantly stimulated the formation of nitrite (7.7 ± 1 nanomoles nitrite/mg protein, n = 6) when compared with basal values (4.1 ± 0.7 nanomoles nitrite/mg protein, n = 6), and this effect was clearly attenuated when metipranolol was present (5.2 ± 0.7 nanomoles nitrite/mg protein, n = 6). Moreover, FeCl2/ascorbate failed to stimulate the formation of nitrite (4.4 ± 0.7 nanomoles nitrite/mg protein, n = 6). 
SNP also stimulated TBARS formation in isolated retinas as opposed to retinal homogenates (Tables 2 3) . The SNP-induced stimulation of TBARS in retinas from Wistar rats was significantly blunted by metipranolol and Trolox but not by timolol (Table 2) . SNP-induced stimulation of TBARS in young RCS rat retinas was slightly greater than in retinas from Wistar rats, but decreased with age, with very little TBARS produced by SNP in retinas from 65-day-old animals (Table 3) . The data from the RCS animals clearly suggested that formation of TBARS takes place primarily in photoreceptors. 
Effect of Intraocular Injection of SNP
Injection of SNP into the aqueous humor resulted in the detection (after 3 days) of a small variable number of scattered TUNEL-positive cells that appeared to be present only in the outer nuclear layer of the retina (Fig. 2) . No attempt was made to quantify the number of TUNEL-positive cells, because there was a great deal of variability in the number of labeled nuclei in every viewed section. Injection of SNP into the vitreous humor again only clearly affected photoreceptors, but the number that stained positively for TUNEL was very much greater (Fig. 3) . Again, however, no attempt was made to quantify the number of TUNEL-positive photoreceptors per retina (3 days after SNP injection), because of the variability in the number of TUNEL-positive cells found between different sections of the same retina and also because TUNEL-positive nuclei appeared in patches. The cause of the variability in number of TUNEL-positive cells in different sections of the same retina is thought to reflect the distances between the actual injection site of SNP in the vitreous and various retinal areas, as depicted in Figure 3 . Moreover, because it was impossible for the site of injection in the vitreous always to be the same, we concluded that it would be difficult to categorically state that fewer TUNEL-positive cells exist in retinas from animals co-injected with SNP and metipranolol or Trolox compared with SNP alone, even if it was possible to count the TUNEL-positive cells in the whole of the retina. It thus became apparent that to achieve such an aim unequivocally it is necessary to analyze the whole of the retina, which would require a biochemical procedure rather than the analysis of retinal sections. 
Caspase-3 and Bcl-2-immuorectivities appeared to be absent from retinas into which saline had been injected into the vitreous (Figs. 4 5) . However, in retinas from eyes injected with SNP, caspase-3 and Bcl-2-immunoreactivities were clearly associated with the photoreceptors. Moreover, as in the case of the staining for TUNEL, caspase-3 and Bcl-2-immunoreactivities showed similar variability in retinas from different animals. Although the amount of immunostaining for both antigens appeared slightly reduced in retinas into which metipranolol or Trolox was co-injected with SNP (compared with SNP alone), a definitive conclusion was impossible to reach for the reasons stated earlier. 
Figure 6 shows the immunostaining of ChAT and nNOS 3 days after intravitreal injection of SNP or NMDA. Although the staining for ChAT and nNOS was clearly altered by NMDA, this was not the case for the SNP-treated retina where the ChAT and nNOS immunoreactivities were identical with those in the control untreated retina. (Note that the changes caused by NMDA were more pronounced when treatment extended over a longer period.) 
The results of Western blot examination of extracts from single whole retinas are shown in Figure 7 . These data clearly show alterations in detected Bcl-2, caspase-3, and PARP proteins. Cleaved protein products from both Bcl-2 (30 kDa) and caspase-3 (17–20 kDa) were increased (relative to actin) in retinas treated with SNP, and PARP (116 kDa) levels were also decreased, suggesting breakdown of the native form of this protein. In each case, the SNP influence was clearly blunted when metipranolol was co-injected. Rhodopsin kinase protein, furthermore, was much reduced in retinas treated with SNP alone but to a lesser extent when metipranolol was present. Analyses of single whole retinal extracts for caspase-3 activity by a colorimetric assay (Fig. 8) supported the finding that SNP-induced stimulation of caspase-3 activity was blunted by metipranolol. 
Discussion
The present results strongly support the findings by Ju et al. 15 that SNP selectively activates the apoptotic pathway in photoreceptors of the rat retina. NO is chemically generated from SNP 16 and it, rather than SNP itself, is the likely cause for the activation of the apoptotic pathway associated with photoreceptors. Support for this idea comes from the finding that when SNP is injected into the aqueous chamber, photoreceptor apoptosis occurs. 15 It is likely that NO gas, generated from SNP, diffuses throughout the eye, causing apoptotic photoreceptor death (Fig. 2) , whereas a chemical like SNP is unlikely to reach the retina from the aqueous chamber. 26 27 The mechanism by which NO causes apoptosis of photoreceptors is unclear. One possibility is that it leads to an excessive build-up of cGMP in photoreceptors. 15 Pretreatment of rats with an NOS inhibitor is known to attenuate ischemia-induced photoreceptor death, 6 and cGMP levels are increased by SNP in photoreceptors, 28 supporting such a view. However, NO-induced photoreceptor apoptosis may involve other processes, because NO is a free radical that can therefore react with various intracellular constituents (e.g., superoxide) to produce the highly reactive substance peroxynitrite, which is known to cause cell damage. 3  
In the present study, SNP was predominantly injected into the vitreous rather than the aqueous humor, to increase the number of photoreceptors dying by apoptosis. The assumption made is that photoreceptor apoptosis occurs when SNP is injected into the aqueous and that NO is the active molecule responsible for this. Support for this tenet comes from the observation that SNP caused a measurable stimulation of nitrite formation in retinal homogenates, whereas FeCl2/ascorbate did not. However, both SNP and FeCl2/ascorbate can stimulate the formation of TBARS in retina (Fig. 1A) and brain 20 homogenates, suggesting that they both generate oxygen radical species. Surprisingly, whereas 100 μM SNP and FeCl2 (25 μM)/ascorbate (100 μM) were found to be equally effective at stimulating TBARS in brain homogenates, 20 this was not the case in the retina, where FeCl2/ascorbate had a much stronger influence than did SNP. 
It appears that NO generated from SNP may only affect photoreceptors in the retina, because there was no obvious influence on other cell types. Support for this is documented by the finding that SNP treatment does not affect ChAT and nNOS immunoreactivities when compared with an intravitreal injection of NMDA. The specificity for the effect of SNP or NO on photoreceptors is documented in the in situ studies, where SNP induced breakdown of DNA (TUNEL staining). Furthermore, an alteration in cellular proteins involved in apoptosis (caspase-3, PARP, and Bcl-2) occurred solely in these cells (Figs. 3 4 5) and from studies performed on retinas from different-aged RCS rats. SNP-induced stimulation of lipid peroxidation or TBARS is most pronounced in retinas from the youngest RCS rats compared with retinas from 65-day-old animals. It is known that photoreceptor degeneration in RCS rats begins at ∼20 days of age and progresses rapidly, so that at 65 days of age no photoreceptors remain. 29  
The in vitro studies on retinal membranes and whole retinas support the view that NO released from SNP reacts with photoreceptor constituents and that reactive nitrogen/oxygen species are generated 3 that subsequently cause lipid peroxidation or production of TBARS. Extensive evidence exists to show that reactive nitrogen/oxygen species cause lipid peroxidation and cell death. 30 31 In our earlier study, we showed that SNP activates lipid peroxidation or TBARS in whole-brain membranes, 20 suggesting that brain and photoreceptor membranes have common features that allow them to be attacked by NO, whereas the membranes of retinal cells other than those of the photoreceptors lack these features. One possible way of explaining these findings is that excessive NO can affect constituents within the myelin of the brain and photoreceptors of the retina to cause lipid peroxidation. However, myelin is absent from the rat retina. The characteristics of the SNP-induced stimulation of lipid peroxidation or TBARS in retinal (photoreceptor) membranes are almost identical with those of whole-brain membranes. 20 In both instances, of the several antiglaucoma drugs tested, only metipranolol and its active metabolite desacetylmetipranolol attenuated SNP-induced stimulation of lipid peroxidation. The IC50 for desacetylmetipranolol and metipranolol was, respectively, 6.9 and 25.1 μM for brain 20 and 3.8 and 3.6 μM for retina (Fig. 1B) . In the experiments on the brain homogenates, the incubation time in the lipid peroxidation assay was 30 minutes, whereas in the present experiments it was 45 minutes. This may reflect the apparent differences in the IC50 values for brain and retinal homogenates. The clear similarity, in both brain and retinal membranes, was also reflected by the antioxidant Trolox, which effectively attenuated SNP stimulation of lipid peroxidation in both tissues. 
When the β-blocker metipranolol is applied topically, it is metabolized to desacetylmetipranolol. This enables it to reduce elevated intraocular pressure in the ocular hypertensive or glaucomatous eye. The fact that metipranolol and its active metabolite can act as antioxidants by attenuating SNP-induced lipid peroxidation indicates that metipranolol has an additional unique property that appears not to be associated with other antiglaucoma substances. This antioxidant property of metipranolol is clearly a positive feature of the compound, for a variety of reasons. For example, should it be possible to administer enough of the substance to reach the retina, it may blunt death processes to photoreceptors and/or ganglion cells in situations where oxidative stress is implicated. It is generally thought that oxidative stress, for example, plays a major part in photoreceptor apoptosis and ganglion cell death in AMD 10 and ischemic syndromes, 32 respectively. Although metipranolol clearly blunts SNP-induced lipid peroxidation in isolated retinas and in retinal membranes, it was still necessary to show that this can occur in vivo. An analysis of retinal sections from retinas where SNP alone or in conjunction with metipranolol (or Trolox) was injected into the vitreous humor for precisely 3 days, however, did not provide clear data. Although intravitreal injection of SNP clearly caused photoreceptor apoptosis (indexed by some of these cells “staining” for TUNEL and caspase-3/Bcl-2 immunoreactivities) the location of this “staining” was variable, making it impossible to quantify reliably. Any attempt to quantify the TUNEL-positive cells would have meant analyzing consecutive sections throughout the whole retina, which was deemed to be too impractical. Variability between the injection site in the vitreous of different animals and the subsequent penetration of the drugs to different parts of the retina makes such an analysis a difficult one. Although a crude analysis of several sections from a single retina suggested that the overall retinal staining for apoptotic markers (TUNEL, caspase-3, Bcl-2) was less when metipranolol or Trolox was co-injected with SNP, it was apparent that definitive data might be obtained only by analysis of the whole of the retina. This was indeed proven to be the case when an analysis was made of whole retinal extracts by a colorimetric assay for caspase-3 and for certain apoptotic proteins (as well as the photoreceptor specific protein, rhodopsin kinase), by electrophoresis/Western blot analysis. The resultant data clearly supported the view that the SNP-induced apoptosis to photoreceptors was blunted by metipranolol (Fig. 7) . The proteins involved in apoptosis, 33 caspase-3 (cleaved, active form) and Bcl-2 (cleaved, active form), were significantly elevated in retinas treated with SNP, and this elevation was blunted when metipranolol was present. Moreover, rhodopsin kinase and the DNA repair enzyme PARP, were much reduced in retinas treated with SNP, and the effect was counteracted when metipranolol was present. The reason that Bcl-2 was elevated may appear to be a contradiction of its proposed role in apoptosis. It is generally understood that Bcl-2 functions to counteract apoptosis, and it is commonly thought to be either downregulated in this death process or not present in sufficient amounts in susceptible cells to prevent death. However, in the untreated retina, Bcl-2 was barely detected, and the possible reason for its increase after SNP treatment is because photoreceptors are being insulted; and, at any one time, the Bcl-2 in some cells is increased as an endogenous protective mechanism (counteracting apoptosis), whereas in others it is downregulated or absent (where apoptosis is induced). Therefore, after 2 days of treatment, the overall amount of Bcl-2 in the retina was elevated in comparison to the control. 
The reason that only one (metipranolol) of the four tested (timolol, carteolol, betaxolol, metipranolol) β-blockers used to treat glaucoma acted as an antioxidant and attenuated SNP-induced photoreceptor apoptosis is unknown but would probably relate to the structure of the molecule. Certain other β-adrenoceptor antagonists, not used in glaucoma, have also been shown to act as antioxidants. 34 35 36 The suggestion has been made that it is the ether linkage between the aromatic moiety and the ethanolamine chain of the typical aryloxypropanolamine structure of β-adrenoceptor antagonists that determines whether a compound is also an antioxidant, 37 since its tautomerization into a phenolic group would allow for the inactivation of free radicals. In addition to this ether linkage, metipranolol possesses an acetyl ester substitution at position 3 of the aromatic ring, which is rapidly desacetylated in vivo yielding a phenolic group that could account for the higher potency of desacetylmetipranolol in inhibiting lipid peroxidation. Under our experimental conditions, it was not possible to establish whether inhibition of lipid peroxidation or photoreceptor apoptosis by metipranolol is mediated entirely by itself or by its partial or total conversion to desacetylmetipranolol. This, however, is of academic interest, given that metipranolol is completely converted in vivo to desacetylmetipranolol, its active metabolite. 
One reason that all antiglaucoma drugs were screened for their capacity to act as antioxidants is because of the existence of evidence from animal studies that such drugs, when topically applied, can reach the retina (Dahlin DC, et al. IOVS 2000;41:ARVO Abstract 2710). 38 39 Any drug reaching the retina may have positive or negative effects within this tissue. Because experimental evidence shows antioxidants have a positive influence on cell death, 31 32 40 41 42 43 44 it must therefore be concluded that should metipranolol reach the retina in glaucoma patients, for example, it is more likely to have a positive effect on visual field loss than would other antiglaucoma drugs. Also, long-term use of metipranolol could prove beneficial in various other ocular diseases related to oxidative stress, such as AMD. 10 The cause of photoreceptor death in AMD, for example, may be similar to that which was demonstrated in the present study in the rat: oxidative stress-induced photoreceptor injury caused by generation of NO. If this is the case, then the present findings may be of clinical relevance where metipranolol is shown to attenuate such an oxidative-induced death to photoreceptors. 
In conclusion, the present study showed that the nonselective β-adrenoceptor antagonist metipranolol and its active metabolite, desacetylmetipranolol, inhibited SNP or NO-induced lipid peroxidation in rat retinal photoreceptors. Moreover, intravitreal injection of SNP caused retinal photoreceptors to specifically reveal characteristics associated with apoptosis, and these characteristics were attenuated when metipranolol was co-injected with SNP. Furthermore, only metipranolol of all antiglaucoma agents studied revealed antioxidant properties and was able to counteract the NO-induced apoptosis. Given the role of free radicals in the pathogenesis of various detrimental processes, the remarkable antioxidant activity of metipranolol and desacetylmetipranolol could be of general clinical relevance. 
 
Figure 1.
 
(a) SNP caused an increase in the production of TBARS in retinal homogenates in a dose-dependent manner (number of individual determinations is shown on appropriate histograms). Also shown, for comparative purposes, is the effect of 25 μM FeCl2/100 μM ascorbate on TBARS production in retinal homogenates. It is clear that FeCl2/ascorbate is much more effective than 100 μM SNP at stimulating TBARS. (b) Comparative effects of different concentrations of metipranolol and desacetylmetipranolol in inhibiting the stimulation of TBARS formation caused by 100 μM SNP.
Figure 1.
 
(a) SNP caused an increase in the production of TBARS in retinal homogenates in a dose-dependent manner (number of individual determinations is shown on appropriate histograms). Also shown, for comparative purposes, is the effect of 25 μM FeCl2/100 μM ascorbate on TBARS production in retinal homogenates. It is clear that FeCl2/ascorbate is much more effective than 100 μM SNP at stimulating TBARS. (b) Comparative effects of different concentrations of metipranolol and desacetylmetipranolol in inhibiting the stimulation of TBARS formation caused by 100 μM SNP.
Table 1.
 
Effect of Antiglaucoma Drugs and Other Compounds on 100 μM SNP-Induced Lipid Peroxidation in Retinal Homogenates
Table 1.
 
Effect of Antiglaucoma Drugs and Other Compounds on 100 μM SNP-Induced Lipid Peroxidation in Retinal Homogenates
Compound Tested TBARS (% SNP alone)
10 μm Metipranolol 42 ± 6*
100 μm Metipranolol 15 ± 3*
10 μm Desacetylmetipranolol 33 ± 3*
100 μm Desacetylmetipranolol 10 ± 2*
100 μm Carteolol 98 ± 7
100 μm Timolol 103 ± 8
100 μm Levobetaxolol 97 ± 9
100 μm Brimonidine 93 ± 11
100 μm Dorzolamide 104 ± 4
10 μm Latanoprost 97 ± 8
10 μm Travoprost 93 ± 7
100 μm Pilocarpine 100 ± 6
10 μm Trolox 32 ± 6*
Table 2.
 
SNP (50 μm) Stimulation of TBARS in Single Albino Rat Retinas
Table 2.
 
SNP (50 μm) Stimulation of TBARS in Single Albino Rat Retinas
Substance TBARS Per Retina (Nanomoles)
SNP 5.9 ± 0.4
SNP + 100 μm metipranolol 0.8 ± 0.1*
SNP + 50 μm metipranolol 1.7 ± 0.2*
SNP + 10 μm metipranolol 3.1 ± 0.3*
SNP + 100 μm timolol 5.7 ± 0.3
SNP + 10 μm trolox 2.0 ± 0.1*
Table 3.
 
SNP (50 μm) Stimulation of TBARS in Single Dystrophic RCS Rat Retinas
Table 3.
 
SNP (50 μm) Stimulation of TBARS in Single Dystrophic RCS Rat Retinas
Age of Animal TBARS per Retina (Nanomoles)
18 Days 8.2 ± 0.7
28 Days 6.1 ± 0.6
50 Days 2.1 ± 0.3
65 Days 0.8 ± 0.1
Figure 2.
 
TUNEL-positive cells (small arrows) were exclusively in the outer nuclear layer after injection of SNP into the aqueous humor. The number of TUNEL-positive cells varied from section to section. (a, b) Two sections are shown that contained quite a few TUNEL-positive cells but many of the sections had very few such cells. Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 2.
 
TUNEL-positive cells (small arrows) were exclusively in the outer nuclear layer after injection of SNP into the aqueous humor. The number of TUNEL-positive cells varied from section to section. (a, b) Two sections are shown that contained quite a few TUNEL-positive cells but many of the sections had very few such cells. Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 3.
 
The variability of the number of TUNEL-positive cells in six retinal sections where SNP was injected into the vitreous humor. The number of TUNEL-positive cells in any one section probably reflects the distance away from the injection site, as shown by the central inset. Almost all the cells in the outer nuclear layer were TUNEL positive (a, b); these sections originated from retinal areas closest to the site of the injected SNP. Sections from other areas farther away from the injected site show fewer TUNEL-positive cells (cf). Many sections showed a distinctly characteristic feature of columns (open arrows) of TUNEL-positive cells (ce). Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 3.
 
The variability of the number of TUNEL-positive cells in six retinal sections where SNP was injected into the vitreous humor. The number of TUNEL-positive cells in any one section probably reflects the distance away from the injection site, as shown by the central inset. Almost all the cells in the outer nuclear layer were TUNEL positive (a, b); these sections originated from retinal areas closest to the site of the injected SNP. Sections from other areas farther away from the injected site show fewer TUNEL-positive cells (cf). Many sections showed a distinctly characteristic feature of columns (open arrows) of TUNEL-positive cells (ce). Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 4.
 
Variable localization of caspase-3 immunoreactivity in three retinal sections (ac) where SNP was injected into the vitreous humor. As with the localization of TUNEL-positive cells caspase-3 immunoreactivity appeared to be in large areas of the outer nuclear layer (a) or in columns of cells (b, c) in this area (open arrows). SNP did not induce staining for caspase-3 immunoreactivity in areas other than the outer nuclear layer. Moreover, caspase-3 immunoreactivity was completely absent from the outer nuclear layer in retinas where vehicle had been injected into the vitreous humor (d). The “staining” in the other parts of the retina was due to the secondary antibody’s recognizing an antigen associated with a retinal blood vessel (small arrows). Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 4.
 
Variable localization of caspase-3 immunoreactivity in three retinal sections (ac) where SNP was injected into the vitreous humor. As with the localization of TUNEL-positive cells caspase-3 immunoreactivity appeared to be in large areas of the outer nuclear layer (a) or in columns of cells (b, c) in this area (open arrows). SNP did not induce staining for caspase-3 immunoreactivity in areas other than the outer nuclear layer. Moreover, caspase-3 immunoreactivity was completely absent from the outer nuclear layer in retinas where vehicle had been injected into the vitreous humor (d). The “staining” in the other parts of the retina was due to the secondary antibody’s recognizing an antigen associated with a retinal blood vessel (small arrows). Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 5.
 
Variable localization of Bcl-2 immunoreactivity in three sections (ac) where SNP was injected into the vitreous humor. As with the localization of TUNEL-positive cells and caspase-3 immunoreactivity, Bcl-2 staining was in large areas (a, b) or in columns (c, open arrows), exclusively in the outer nuclear layer. Bcl-2 immunoreactivity was absent from the outer nuclear layer (d) where vehicle was injected into the vitreous humor. Any staining in the inner parts of the retina (small arrows) was due to the secondary antibody’s action on retinal vessels. Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 5.
 
Variable localization of Bcl-2 immunoreactivity in three sections (ac) where SNP was injected into the vitreous humor. As with the localization of TUNEL-positive cells and caspase-3 immunoreactivity, Bcl-2 staining was in large areas (a, b) or in columns (c, open arrows), exclusively in the outer nuclear layer. Bcl-2 immunoreactivity was absent from the outer nuclear layer (d) where vehicle was injected into the vitreous humor. Any staining in the inner parts of the retina (small arrows) was due to the secondary antibody’s action on retinal vessels. Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 6.
 
ChAT (ac) and nNOS (df) immunoreactivities in the retina (a, d) and after intravitreal injection of either SNP (b, e) or NMDA (c, f). ChAT immunoreactivity was associated with a population of amacrine cells; their perikarya (thick arrows) being on either side of two clear laminae (thin arrows) of terminals in the inner plexiform layer (a). Small arrows: staining of retinal blood vessels caused by the secondary antibody (ac). It can be seen that the normal ChAT immunoreactivity is unaffected by SNP (b) but not by NMDA (c). Injection of NMDA resulted in fewer ChAT-positive perikarya, and the normal double layer of terminals in the inner plexiform layer was reduced to a single layer (c). NOS-immunoreactivity in the control retina (d) appeared as three large diffuse bands in the inner plexiform layer (three thin parallel arrows) and perikarya (large short arrow) that were few in number. The normal NOS-immunoreactivity was unaffected by SNP (e) but was almost completely reduced to a thin band after NMDA injection (f, large arrow). Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 6.
 
ChAT (ac) and nNOS (df) immunoreactivities in the retina (a, d) and after intravitreal injection of either SNP (b, e) or NMDA (c, f). ChAT immunoreactivity was associated with a population of amacrine cells; their perikarya (thick arrows) being on either side of two clear laminae (thin arrows) of terminals in the inner plexiform layer (a). Small arrows: staining of retinal blood vessels caused by the secondary antibody (ac). It can be seen that the normal ChAT immunoreactivity is unaffected by SNP (b) but not by NMDA (c). Injection of NMDA resulted in fewer ChAT-positive perikarya, and the normal double layer of terminals in the inner plexiform layer was reduced to a single layer (c). NOS-immunoreactivity in the control retina (d) appeared as three large diffuse bands in the inner plexiform layer (three thin parallel arrows) and perikarya (large short arrow) that were few in number. The normal NOS-immunoreactivity was unaffected by SNP (e) but was almost completely reduced to a thin band after NMDA injection (f, large arrow). Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 7.
 
Western blot analyses of whole retinal extracts 3 days after intravitreal injection of 50 μM SNP. Shown are representative blots for actin (a; 45 kDa), caspase-3 (b; 17–20 kDa), PARP (c; 116 kDa), rhodopsin kinase (d; 65 kDa), and Bcl-2 (e; 30 kDa). Also shown are graphic representations of densitometric analyses (n = 3–5 sets of samples) for Western blots obtained for each antigen. Blot images were prepared by scanning labeled nitrocellulose to produce digital images. *P < 0.05, compared with saline alone by unpaired Student’s t-test.
Figure 7.
 
Western blot analyses of whole retinal extracts 3 days after intravitreal injection of 50 μM SNP. Shown are representative blots for actin (a; 45 kDa), caspase-3 (b; 17–20 kDa), PARP (c; 116 kDa), rhodopsin kinase (d; 65 kDa), and Bcl-2 (e; 30 kDa). Also shown are graphic representations of densitometric analyses (n = 3–5 sets of samples) for Western blots obtained for each antigen. Blot images were prepared by scanning labeled nitrocellulose to produce digital images. *P < 0.05, compared with saline alone by unpaired Student’s t-test.
Figure 8.
 
Measurement of caspase-3 activity in whole retinal homogenates 3 days after intravitreal injection of 50 μM SNP. The assay assessed the amount of caspase activity present in samples that specifically recognized the peptide substrate DEVD. Results are expressed as amount of colorimetric label pNA cleaved from synthetic DEVD per retina during the assay period (n = 4 for each determination). *P < 0.05, by unpaired Student’s t-test. No significant difference between saline and saline plus metipranolol.
Figure 8.
 
Measurement of caspase-3 activity in whole retinal homogenates 3 days after intravitreal injection of 50 μM SNP. The assay assessed the amount of caspase activity present in samples that specifically recognized the peptide substrate DEVD. Results are expressed as amount of colorimetric label pNA cleaved from synthetic DEVD per retina during the assay period (n = 4 for each determination). *P < 0.05, by unpaired Student’s t-test. No significant difference between saline and saline plus metipranolol.
The authors thank Helmut Allmeier (Dr. Mann Pharma, Berlin, Germany) for support and for supplying des acetylmetipranolol. 
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Figure 1.
 
(a) SNP caused an increase in the production of TBARS in retinal homogenates in a dose-dependent manner (number of individual determinations is shown on appropriate histograms). Also shown, for comparative purposes, is the effect of 25 μM FeCl2/100 μM ascorbate on TBARS production in retinal homogenates. It is clear that FeCl2/ascorbate is much more effective than 100 μM SNP at stimulating TBARS. (b) Comparative effects of different concentrations of metipranolol and desacetylmetipranolol in inhibiting the stimulation of TBARS formation caused by 100 μM SNP.
Figure 1.
 
(a) SNP caused an increase in the production of TBARS in retinal homogenates in a dose-dependent manner (number of individual determinations is shown on appropriate histograms). Also shown, for comparative purposes, is the effect of 25 μM FeCl2/100 μM ascorbate on TBARS production in retinal homogenates. It is clear that FeCl2/ascorbate is much more effective than 100 μM SNP at stimulating TBARS. (b) Comparative effects of different concentrations of metipranolol and desacetylmetipranolol in inhibiting the stimulation of TBARS formation caused by 100 μM SNP.
Figure 2.
 
TUNEL-positive cells (small arrows) were exclusively in the outer nuclear layer after injection of SNP into the aqueous humor. The number of TUNEL-positive cells varied from section to section. (a, b) Two sections are shown that contained quite a few TUNEL-positive cells but many of the sections had very few such cells. Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 2.
 
TUNEL-positive cells (small arrows) were exclusively in the outer nuclear layer after injection of SNP into the aqueous humor. The number of TUNEL-positive cells varied from section to section. (a, b) Two sections are shown that contained quite a few TUNEL-positive cells but many of the sections had very few such cells. Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 3.
 
The variability of the number of TUNEL-positive cells in six retinal sections where SNP was injected into the vitreous humor. The number of TUNEL-positive cells in any one section probably reflects the distance away from the injection site, as shown by the central inset. Almost all the cells in the outer nuclear layer were TUNEL positive (a, b); these sections originated from retinal areas closest to the site of the injected SNP. Sections from other areas farther away from the injected site show fewer TUNEL-positive cells (cf). Many sections showed a distinctly characteristic feature of columns (open arrows) of TUNEL-positive cells (ce). Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 3.
 
The variability of the number of TUNEL-positive cells in six retinal sections where SNP was injected into the vitreous humor. The number of TUNEL-positive cells in any one section probably reflects the distance away from the injection site, as shown by the central inset. Almost all the cells in the outer nuclear layer were TUNEL positive (a, b); these sections originated from retinal areas closest to the site of the injected SNP. Sections from other areas farther away from the injected site show fewer TUNEL-positive cells (cf). Many sections showed a distinctly characteristic feature of columns (open arrows) of TUNEL-positive cells (ce). Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 4.
 
Variable localization of caspase-3 immunoreactivity in three retinal sections (ac) where SNP was injected into the vitreous humor. As with the localization of TUNEL-positive cells caspase-3 immunoreactivity appeared to be in large areas of the outer nuclear layer (a) or in columns of cells (b, c) in this area (open arrows). SNP did not induce staining for caspase-3 immunoreactivity in areas other than the outer nuclear layer. Moreover, caspase-3 immunoreactivity was completely absent from the outer nuclear layer in retinas where vehicle had been injected into the vitreous humor (d). The “staining” in the other parts of the retina was due to the secondary antibody’s recognizing an antigen associated with a retinal blood vessel (small arrows). Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 4.
 
Variable localization of caspase-3 immunoreactivity in three retinal sections (ac) where SNP was injected into the vitreous humor. As with the localization of TUNEL-positive cells caspase-3 immunoreactivity appeared to be in large areas of the outer nuclear layer (a) or in columns of cells (b, c) in this area (open arrows). SNP did not induce staining for caspase-3 immunoreactivity in areas other than the outer nuclear layer. Moreover, caspase-3 immunoreactivity was completely absent from the outer nuclear layer in retinas where vehicle had been injected into the vitreous humor (d). The “staining” in the other parts of the retina was due to the secondary antibody’s recognizing an antigen associated with a retinal blood vessel (small arrows). Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 5.
 
Variable localization of Bcl-2 immunoreactivity in three sections (ac) where SNP was injected into the vitreous humor. As with the localization of TUNEL-positive cells and caspase-3 immunoreactivity, Bcl-2 staining was in large areas (a, b) or in columns (c, open arrows), exclusively in the outer nuclear layer. Bcl-2 immunoreactivity was absent from the outer nuclear layer (d) where vehicle was injected into the vitreous humor. Any staining in the inner parts of the retina (small arrows) was due to the secondary antibody’s action on retinal vessels. Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 5.
 
Variable localization of Bcl-2 immunoreactivity in three sections (ac) where SNP was injected into the vitreous humor. As with the localization of TUNEL-positive cells and caspase-3 immunoreactivity, Bcl-2 staining was in large areas (a, b) or in columns (c, open arrows), exclusively in the outer nuclear layer. Bcl-2 immunoreactivity was absent from the outer nuclear layer (d) where vehicle was injected into the vitreous humor. Any staining in the inner parts of the retina (small arrows) was due to the secondary antibody’s action on retinal vessels. Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 6.
 
ChAT (ac) and nNOS (df) immunoreactivities in the retina (a, d) and after intravitreal injection of either SNP (b, e) or NMDA (c, f). ChAT immunoreactivity was associated with a population of amacrine cells; their perikarya (thick arrows) being on either side of two clear laminae (thin arrows) of terminals in the inner plexiform layer (a). Small arrows: staining of retinal blood vessels caused by the secondary antibody (ac). It can be seen that the normal ChAT immunoreactivity is unaffected by SNP (b) but not by NMDA (c). Injection of NMDA resulted in fewer ChAT-positive perikarya, and the normal double layer of terminals in the inner plexiform layer was reduced to a single layer (c). NOS-immunoreactivity in the control retina (d) appeared as three large diffuse bands in the inner plexiform layer (three thin parallel arrows) and perikarya (large short arrow) that were few in number. The normal NOS-immunoreactivity was unaffected by SNP (e) but was almost completely reduced to a thin band after NMDA injection (f, large arrow). Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 6.
 
ChAT (ac) and nNOS (df) immunoreactivities in the retina (a, d) and after intravitreal injection of either SNP (b, e) or NMDA (c, f). ChAT immunoreactivity was associated with a population of amacrine cells; their perikarya (thick arrows) being on either side of two clear laminae (thin arrows) of terminals in the inner plexiform layer (a). Small arrows: staining of retinal blood vessels caused by the secondary antibody (ac). It can be seen that the normal ChAT immunoreactivity is unaffected by SNP (b) but not by NMDA (c). Injection of NMDA resulted in fewer ChAT-positive perikarya, and the normal double layer of terminals in the inner plexiform layer was reduced to a single layer (c). NOS-immunoreactivity in the control retina (d) appeared as three large diffuse bands in the inner plexiform layer (three thin parallel arrows) and perikarya (large short arrow) that were few in number. The normal NOS-immunoreactivity was unaffected by SNP (e) but was almost completely reduced to a thin band after NMDA injection (f, large arrow). Images were prepared as photographs and scanned to produce digital images. Scale bars, 40 μm.
Figure 7.
 
Western blot analyses of whole retinal extracts 3 days after intravitreal injection of 50 μM SNP. Shown are representative blots for actin (a; 45 kDa), caspase-3 (b; 17–20 kDa), PARP (c; 116 kDa), rhodopsin kinase (d; 65 kDa), and Bcl-2 (e; 30 kDa). Also shown are graphic representations of densitometric analyses (n = 3–5 sets of samples) for Western blots obtained for each antigen. Blot images were prepared by scanning labeled nitrocellulose to produce digital images. *P < 0.05, compared with saline alone by unpaired Student’s t-test.
Figure 7.
 
Western blot analyses of whole retinal extracts 3 days after intravitreal injection of 50 μM SNP. Shown are representative blots for actin (a; 45 kDa), caspase-3 (b; 17–20 kDa), PARP (c; 116 kDa), rhodopsin kinase (d; 65 kDa), and Bcl-2 (e; 30 kDa). Also shown are graphic representations of densitometric analyses (n = 3–5 sets of samples) for Western blots obtained for each antigen. Blot images were prepared by scanning labeled nitrocellulose to produce digital images. *P < 0.05, compared with saline alone by unpaired Student’s t-test.
Figure 8.
 
Measurement of caspase-3 activity in whole retinal homogenates 3 days after intravitreal injection of 50 μM SNP. The assay assessed the amount of caspase activity present in samples that specifically recognized the peptide substrate DEVD. Results are expressed as amount of colorimetric label pNA cleaved from synthetic DEVD per retina during the assay period (n = 4 for each determination). *P < 0.05, by unpaired Student’s t-test. No significant difference between saline and saline plus metipranolol.
Figure 8.
 
Measurement of caspase-3 activity in whole retinal homogenates 3 days after intravitreal injection of 50 μM SNP. The assay assessed the amount of caspase activity present in samples that specifically recognized the peptide substrate DEVD. Results are expressed as amount of colorimetric label pNA cleaved from synthetic DEVD per retina during the assay period (n = 4 for each determination). *P < 0.05, by unpaired Student’s t-test. No significant difference between saline and saline plus metipranolol.
Table 1.
 
Effect of Antiglaucoma Drugs and Other Compounds on 100 μM SNP-Induced Lipid Peroxidation in Retinal Homogenates
Table 1.
 
Effect of Antiglaucoma Drugs and Other Compounds on 100 μM SNP-Induced Lipid Peroxidation in Retinal Homogenates
Compound Tested TBARS (% SNP alone)
10 μm Metipranolol 42 ± 6*
100 μm Metipranolol 15 ± 3*
10 μm Desacetylmetipranolol 33 ± 3*
100 μm Desacetylmetipranolol 10 ± 2*
100 μm Carteolol 98 ± 7
100 μm Timolol 103 ± 8
100 μm Levobetaxolol 97 ± 9
100 μm Brimonidine 93 ± 11
100 μm Dorzolamide 104 ± 4
10 μm Latanoprost 97 ± 8
10 μm Travoprost 93 ± 7
100 μm Pilocarpine 100 ± 6
10 μm Trolox 32 ± 6*
Table 2.
 
SNP (50 μm) Stimulation of TBARS in Single Albino Rat Retinas
Table 2.
 
SNP (50 μm) Stimulation of TBARS in Single Albino Rat Retinas
Substance TBARS Per Retina (Nanomoles)
SNP 5.9 ± 0.4
SNP + 100 μm metipranolol 0.8 ± 0.1*
SNP + 50 μm metipranolol 1.7 ± 0.2*
SNP + 10 μm metipranolol 3.1 ± 0.3*
SNP + 100 μm timolol 5.7 ± 0.3
SNP + 10 μm trolox 2.0 ± 0.1*
Table 3.
 
SNP (50 μm) Stimulation of TBARS in Single Dystrophic RCS Rat Retinas
Table 3.
 
SNP (50 μm) Stimulation of TBARS in Single Dystrophic RCS Rat Retinas
Age of Animal TBARS per Retina (Nanomoles)
18 Days 8.2 ± 0.7
28 Days 6.1 ± 0.6
50 Days 2.1 ± 0.3
65 Days 0.8 ± 0.1
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