Modulation of Retinal Dopaminergic Cells by Nitric Oxide. a Protective Effect on NMDA-Induced Retinal Injury

in vivo 18: 311-316 (2004)

Review Modulation of Retinal Dopaminergic Cells by Nitric Oxide. A Protective Effect on NMDA-induced Retinal Injury

YASUSHI KITAOKA1 and TOSHIO KUMAI2

Departments of 1Ophthalmology and 2Pharmacology, St Marianna University School of Medicine, Kawasaki, Kanagawa 216-8511, Japan

Abstract. Nitric oxide (NO) may play an important role in experimental glaucoma (7). Thus, the involvement of regulating retinal neuronal survival. While the precise impact of elevated vitreal glutamate in glaucoma is not well NO mechanisms on retinal neurons remains to be elucidated, it established. Nonetheless, an understanding of the has been reported that low doses of NO may have a biochemical mechanism by which glutamate regulates neuroprotective effect against N-methyl-D-aspartate (NMDA)- retinal neuronal survival is important in developing induced retinal neurotoxicity. Dopamine has also been therapeutics for protecting retinal neuronal cells at the recognized to have neuroprotective actions. Retinal dopaminergic injured site in several ocular disorders including glaucoma, cells can be detected by the immunohistochemical staining of ischemia (8,9) and optic neuropathy (10,11). tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. We observed that NMDA dramatically decreased in TH NMDA-induced neurotoxicity immunostaining at the junction between the inner nuclear layer and inner plexiform layer, and that this reduction in The excitotoxic effect of glutamate on the retinal neuronal immunostaining was attenuated by the co-injection of NOC 18, cells has been well demonstrated. Among the several an NO donor. Thus, the current review focused on the NO- glutamate receptors, activation of the N-methyl-D-aspartate dopamine interactions in the retinal neuroprotection. (NMDA) receptor causes the degeneration of retinal neuronal cells (12,13). The overstimulation of NMDA Glaucomatous optic damage is associated with the death of receptors initiates a cascade of events involving necrosis and retinal ganglion cells (RGCs). It has been reported that apoptosis in vitro (14). In an in vivo system, Siliprandi et al. large RGCs may be selectively damaged in glaucoma (1-3), (15) demonstrated that a single intravitreal injection of whereas certain types of glaucoma are associated with NMDA led to a loss of cells in the ganglion cell layer (GCL) elevated glutamate in the vitreous body (4). In addition, a and a reduction in the thickness of the inner plexiform layer chronic low-dose elevation of intravitreal glutamate levels (IPL) in the rat retina. Lam et al. reported that TUNEL- can induce RGC death (5). Furthermore, glutamate causes positive cells appeared in the GCL and inner nuclear layer preferentially larger RGC death (6). Therefore, it has been (INL) following an NMDA intravitreal injection. It has suggested that glutamate may play an important role in recently been reported that the intravitreal injection of glaucoma. However, recent studies have reported that NMDA activates a p53-dependent pathway of death in the vitreal glutamate concentrations were not elevated in RGCs of adult mice (16). The involvement of p53 in monkey eyes with anatomic and functional damage from regulating apoptosis in a variety of neuronal cell types is widely known. Furthermore, Manabe and Lipton (17) suggested that NMDA stimulation leads to both proapoptotic, p38 mitogen-activated protein kinase and Correspondence to: Yasushi Kitaoka, Department of antiapoptotic, phosphatidylinositol-3 kinase-Akt, pathways Ophthalmology, St Marianna University School of Medicine, 2-16- in the retina. Thus, even in vivo, several molecular events, 1 Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan. Tel: 81-44-977-8111, Fax: 81-44-976-0509, e-mail: [email protected] including an apoptotic pathway, may play an important role in NMDA-induced retinal neurotoxicity. With respect to Key Words: Dopaminergic cells, nitric oxide, N-methyl-D-aspartate, another mechanism, NMDA-induced retinal damage is also retina, tyrosine hydroxylase. associated with the activation of nitric oxide synthase

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Figure 1. Immunohistochemistry for tyrosine hydroxylase (TH) of the rat retina at 5 days following intravitreal injection of phosphate-buffered saline (PBS), 200 nmol N-methyl-D-aspartate (NMDA), or 200 nmol NMDA + 0.5 nmol 1-hydroxy-2-oxo-3,3-bis (2-aminoethyl)-1-triazene (NOC 18). (A) Abundant TH immunostaining was observed in the area between the inner nuclear layer (INL) and the inner plexiform layer (IPL) in the control retina (A, arrowhead). The arrow indicates the dopaminergic cell located in the innermost INL. (B) No immunostaining was observed in the NMDA-treated retina. (C) Some TH immunostaining can be observed in the NMDA-treated retina with the co-injection of NOC 18 (C, arrowheads). Scale bar = 50 Ìm.

(NOS), which results in nitric oxide (NO) production (18), ganglion cells may have a postjunctional function in the whereas it was reported that the NMDA receptor effect was dendrites and somas of the retinal ganglion cells and a diminished by L-arginine, a natural substrate of NOS (19). neuromodulator function at the nerve terminals of the retinal In addition, it has been reported that NO can decrease ganglion cells (29). Furthermore, NO produced by NOS in the NMDA receptor activity (20,21). Thus, it is likely that amacrine and ganglion cells may be a paracrine modulator of NMDA and NO may influence each other. cell death within the retinal tissue (30).

Localization of NOS in the retina Possible role of NO in the retina

NO is generated by the oxidation of L-arginine, a reaction that NO, a free radical gas with a half-life of a few seconds, has is catalyzed by the enzyme NOS (22). Several investigators been shown to play various physiological and reported the expression of NOS in the retina (23-29). NADPH- pathophysiological roles in the nervous system (31). NO has diaphorase staining has been used as a histological marker of been reported to have both neuroprotective (20,32) and NOS (23-27). NOS-expressing neurons were found in both the neurotoxic functions (33,34) in the central nervous system INL and GCL (24) and two types of amacrine cells in the INL (CNS). NO may also play a controversial role in retinal containing NOS have also been distinguished (25,26). In adult neuronal cell death (18,35,36). One possible cause of this rat retina, weakened immunoreactivity of the endothelial NOS role is the redox state of NO (37). Lipton et al. (37) (eNOS) protein was observed in the IPL and the contour of suggested that NO might lead to neurotoxicity when it large cells was stained within the GCL (28). Furthermore, the reacts with superoxide, but it prevents NMDA receptor- immunoreactivity of the neuronal NOS (nNOS) protein in the mediated neurotoxicity when converted to NO+. Another IPL was weakened and the contour of large cells was stained possibility is that this role is due to the different within the GCL (28). nNOS mRNA is present in many cell experimental concentrations of NO. Kashii et al. (18) bodies in the INL and is present in increased amounts in large reported that low concentrations (50 ÌM) of S- cell bodies proximal to the IPL in the retina of rats (29). In the nitrosocysteine (SNOC; an NO-generating agent) or sodium GCL, cells with large somas are positive for nNOS mRNA (29). nitroprusside (SNP; an NO donor) had neuroprotective In human eyes, the nNOS mRNA and nNOS proteins were effects on retinal neurons against NMDA-induced also present in the INL and GCL (29). The NOS in retinal neurotoxicity. However, high concentrations (500 ÌM) of

312 Kitaoka and Kumai: Modulation of Retinal Dopaminergic Cells by NO

SNOC or SNP alone both caused a reduction in cell region (43,44). Pilz et al. (45) demonstrated that cGMP and viability. Furthermore, Kashiwagi et al. (35) reported that NO activate JunB, c-Jun, JunD and c-Fos mRNA NO has a dual action on retinal RGC survival. At ≥ 100 transcriptions. JunB, c-Jun, JunD and c-Fos comprise the ÌM, S-nitroso-N-acetylpenicillamine (an NO donor) AP-1 complex. In addition, Canals et al. (46) reported that significantly reduced RGC survival in a concentration- low concentrations of NO increased the number of TH dependent manner, whereas low concentrations increased immunoreactive cells and the TH protein levels in fetal RGC survival, particularly that of large RGCs, in vitro. midbrain cultures. These findings are supported by recent Thus, it has been suggested that, at low concentrations, NO studies which indicated that NO prevents NMDA-induced may be neuroprotective of retinal neuronal cells. reduction in the TH mRNA and TH protein levels in the In an in vivo system, we previously reported that 0.5 nmol retina (38). Whether NO regulates the TH expression at the of 1-hydroxy-2-oxo-3,3-bis (2-aminoethyl)-1-triazene (NOC transcriptional level in the retina remains to be elucidated; 18; an NO donor) significantly attenuated NMDA-induced however, it is suggested that low concentrations of NO may neurotoxicity in the rat retina (38). Conversely, Takahata et al. be neuroprotective of retinal dopaminergic cells. (39) reported that 1-hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)- 3-ethyl-1-triazene (NOC 12; an NO donor) induced dose- Possible role of dopamine in the retina dependent (over 200 nmol) retinal neurotoxicity. The half-life of NO release of NOC 18 is 21 h and that of NOC 12 is 100 Dopamine is a retinal neurotransmitter that regulates min (37ÆC, pH7.4). It has recently been reported that a short- several functions of various cells, including photoreceptors, lifetime NO donor, such as NOC 12, causes greater retinal horizontal cells, bipolar cells and ganglion cells (47). damage than a long-lifetime NO donor, NOC 18 (40). Dopaminergic receptors that mediate these actions of Therefore, the regulation of retinal neuronal cell survival by dopamine are generally classified into two groups, D1-like NO may be related not only to the NO concentration of the and D2-like receptors. It was shown that the D1 receptors working stock but also to its intravitreal retention time. were localized predominantly in the inner retina, including the IPL and INL (48), probably in horizontal and cone Retinal dopaminergic cells bipolar cells (49). Conversely, the D2 receptors were localized predominantly in the photoreceptor inner and In the rat retina, dopaminergic cells can be detected by the outer segments and in the outer nuclear layer (48). immunohistochemical staining of tyrosine hydroxylase (TH), Furthermore, it has been suggested that retinal the rate-limiting enzyme in dopamine synthesis. The dopaminergic cells, which may release dopamine, possess majority of TH immunoreactive cells are located in the presynaptic D2 receptors and that the stimulation of these innermost sublayer of the INL and their dendrites ramify at receptors may inhibit the synthesis and release of retinal the junction between the INL and IPL (Figure 1A). These dopamine (47). Thus, the D1 and D2 receptors were results are consistent with those of a previous report in the localized at different sites and perform different actions. rat retina (41). We previously found that NMDA causes Dopamine can inhibit NMDA receptor activity in significant decreases in the expression of TH mRNA and neurons from the chick retina (50). Furthermore, it was the production of TH protein (38). Consistent with these shown that dopamine protects cultured rat retinal neurons results, immunohistochemical studies demonstrated that the against NMDA receptor-mediated glutamate neurotoxicity intravitreal injection of 200 nmol NMDA dramatically through the dopamine D1 receptors (51). We previously decreased the TH immunostaining at 5 days after injection found that NMDA reduced the dopamine levels of the (Figure 1B). In CNS, it was shown that NMDA decreased retina and that this decrease in the level of dopamine was the number of TH immunoreactive cells in the cultures of attenuated by NO (38). By means of a voltammetric study, mesencephalic cells (42). These findings indicate that Iravani et al. (52) reported that the pressure injection of NO dopaminergic cells in the retina are vulnerable to NMDA- into rat caudate putamen slices caused the release of dose- induced injury. dependent dopamine. Moreover, other reports have shown We also observed that 0.5 nmol of NOC 18 attenuated that NO increases the striatal dopamine release in vivo NMDA-induced decreases in TH immunostaining of the rat (53,54). Thus, NO may also affect the level of dopamine in retina (Figure 1C). This suggests the possibility that low the retina in addition to the TH expression. Since TH is the concentrations of NO can protect the dopaminergic cells rate-limiting enzyme in dopamine synthesis, an increase in against NMDA-induced injury and/or that NO regulates the the TH level results in an increase in the dopamine level. TH protein production of dopaminergic cells. It has been Therefore, taking together the above findings, it is likely suggested that NO increases the TH transcriptional activity. that low doses of NO results in an increment in the TH AP-1 binding domain and cyclic AMP responsive elements expression and an increase in the dopamine level in the are present in the TH gene transcription enhancer/promoter retina.

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