Nitric oxide in the eye: Multifaceted roles and diverse outcomes

SURVEY OF OPHTHALMOLOGY

VOLUME

42

l

NUMBER

I . JULY-AUGUST

1997

ELSEVIER

CURRENT RESEARCH EDWARD COTLIER

AND ROBERT WEINREB, EDITORS

Nitric Oxide in the Eye: Multifaceted Diverse Outcomes F. BECQUET,

MD, PHD, Y. COURTOIS,

Roles and

PI-ID, AND 0. GOUREAU,

PHD

Retinal Development, Aging, and Pathology Laboratory, Inserm U450, Claude Bernard Association, University of Paris V, Paris, France

Abstract. Recent works have highlighted the role of nitric oxide in a wide array of disease entities, including septic shock, hypertension, cerebral ischemia, and chronic degenerative diseases of the nervous system. The functions of nitric oxide appear very diverse, having actions on vascular tone, neurotransmission, immune cytotoxicity, and many others. Nitric oxide is an important mediator of homeostatic processes in the eye, such as regulation of aqueous humor dynamics, retinal neurotransmission and phototransduction. Changes in its generation or actions could contribute to pathological states such as inflammatory diseases (uveitis, retinitis) or degenerative diseases (glaucoma, retinal degeneration). Localization in the eye and biochemical characteristics of nitric oxide will be reviewed. A better understanding of the nitric oxide pathway will be the key to the development of new approaches to the management and treatment of various ocular diseases. (Surv Ophthalmol42:71-82, 1997. 0 1997 by Elsevier Science Inc. All rights reserved.) Keywords. glaucoma nitric oxide synthase

l l

inflammation l NADPH-diaphorase nitric oxide synthase inhibitors l retina

Nitric oxide, an atmospheric gas, is known to be enzymatically synthesized in a tightly regulated manner by a number of cell types. Nitric oxide is soluble in tissues and freely diffusible across membranes. Over the past 6 years, significant progress has been made in elucidating the mechanism of nitric oxide synthesis and its functions in different biological systems. Nitric oxide serves a wide variety of functions both intracellularly as a second messenger that responds to activation of plasma membrane receptors, and extracellularly as a paracrine factor that carries information between cells. The functions of this free radical appear very diverse, having actions on vascular tone, neurotransmission,g immune cytotoxicity, 50875and many others. The nitric oxide pathway has also been studied in

l

nitric oxide

l

the eye and this review identifies some of the most important physiological and pathophysiological functions of nitric oxide (and the consequences of their inhibition by nitric oxide synthase inhibitors) that may interact to determine outcomes after different ocular pathologies. The potential therapeutic utility of both nitric oxide synthesis inhibitors and nitric oxide donors will be exposed and discussed as new therapeutic approaches in ocular diseases.

I. Characteristics A. BIOSYNTHESIS

OF NITRIC

of Nitric Oxide OXIDE

Nitric oxide is synthetized by the enzyme nitric oxide synthase (NOS) from the oxidation of the guanidino nitrogen of its substrate L-arginine with 71

0 1997 by Elsevier All rights reserved.

Science

Inc.

0039-6257/97/$17.00 PII s0039-6257(97)00013-1

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stochiometric formation of L-citrulline.58~F” Regulation of biosynthesis of nitric oxide is very important since, unlike other neurotransmitters, nitric oxide cannot be stored or released by conventional regulatory mechanisms. Three isoforms of NOS, cloned by three distinct gene products,22,“5@ have been identified as being responsible for nitric oxide synthesis in the presence of oxygen, the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) , and flavins. Two types of the enzyme are continuously present and, thus, are termed constitutive NOS. The first, termed NOSI, is found essentially in some neurons of the central and the peripheral nervous system,” and the second (NOS-III) was originally expressed by the vascular endothelium. “2,45 Small amounts of nitric oxide are generated by these two isoforms when they are activated by the calcium/calmodulin complex (Fig. 1). In contrast, NOS-II, or inducible NOS, is expressed in many cell types after challenge by immunological or inflammatory stimuli50.75 (Fig. 1). This isoform, the activity of which is generally independent of calcium, generates large amounts of nitric oxide over longer periods which are dependent on the presence of the stimuli. The synthesis of nitric oxide can be inhibited by several L-arginine analogues, including No-mono-

Endothelialcells Neurons

A Vasodilatators

I

methyl-Larginine (L-NMh4A) , NG-nitroLarginine methyl ester (L-NAME), and NC-nitroLarginine (LNA) .‘4.‘5 The inhibition of NOS by these substrate analogues can initially be reversed by simultaneous application of excess arginine, consistent with their competitive blockade of the active site. Newer classes of NOS inhibitors, such as thiocitrulline, No-iminoethyl-lysine or nonaminoacid inhibitors, including aminoguanidine, nitroindazole and isothioureas, have been more recently reported and exhibit different specificity for the distinct isoforms of NOS.44.‘” Over the past three years, the nitric oxide pathway has also been studied in the eye, and both the inducible and constitutive isoforms have been identified. Different techniques were used to describe them. Historically, the first one is the NADPHdiaphorase staining, which is almost always associated with NOS immunoreactivity.g~47~66 With the characterization of their sequences, specific polyclonal antibodies have been prepared and have been used to determine the precise localization of the different NOS in the eye. The presence of NOS has been established directly by the evaluation of its enzymatic activity (transformation of radioactive L-arginine to radioactive L-citrulline) in crude tissue prepara-

Smooth muscle Neurons

L-arginine I

1 GTP

I

Glutamate

Pathogenic agents Neighbouringcells

Macrophagesand other numerouscells

B

L-arginine

Endotoxin \

bN0

.mitochondrial

Cytokines /I L-citrulline

Stimulus

Producing cell

ET AL

Target cell

Fig. 1. Nitric oxide interactions with cells after its production by constitutives (NO!%1 and NOSIII) or inducible (NOS-II) NOS. A: Constitutives NOS were found essentially in some neurons of the central and peripheral nervous system (NO.%), and in the vascular endothelium (NOS-III). These two isoforms are activated by the calcium/calmodulin complex after an intracellular increase of calcium (Ca”), and then release small amounts of nitric Oxide, which entail physiological effects after activation of guanylate cyclase (neurotransmission, vasodilatation). (GTP: guanosine triphosphate; cGMP: cyclic guanosine monophosphate). B: Inducible NOS, is expressed in many cell types after challenge by immunological or inflammatory stimuli (cytokines and endotoxin), and release large amounts of nitric oxide over longer periods, which could diffuse and react with different cellular targets.

NITRIC

OXIDE

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IN THE EYE

tion.2”*‘02The subcellular localization and the different cofactors’ requirements, (essentially the calcium dependency), for each form of NOS help to discriminate biochemically type I, II and III.22 Recently, molecular biology techniques, such as reverse transcriptase-polymerase chain reaction (RT-PCR), or in situ hybridization, allow, respectively, a very sensitive quantification or localization of the specific mRNA of each NOS-I, II or III forms.3’,‘2 B. CHEMICAL RJ3ACTIVITY AND MOLECULAR TARGETS OF NITRIC OXIDE

Nitric oxide reacts in biological systemswith oxygen, superoxide anion (O;-) and transition metals, producing nitrite and nitrate, peroxynitrite (ONOO-) and metal-nitric oxide adducts, respectively.” Furthermore, nitric oxide can react with thiols, only after its oxidation to a nitrosating species possessing(NO’) characterq5,“” (Table 1). The physiological amounts of nitric oxide released by NOS-I and NOS-III isoforms are low and participate in nitric oxide signaling pathways in the cardiovascular and neuronal system, while larger amounts of nitric oxide synthesized by NOS-II contribute to the cytotoxic/cytostatic actions (Fig. 1). Some of the actions of nitric oxide (i.e., neurotransmission, vasodilatation) are mediated via the activation of soluble guanylate cyclase and subsequent elevation of cyclic guanosine monophosphate (cGMP) levels.“’ Interaction of nitric oxide with hemoglobin and with other different heme-containing proteins (cyclooxygenase, cytochrome-P450s) was also reported.50 Nitric oxide in target cells has been shown to affect proteins conTABLE Molecular

Targets of Nitric

Targets Heme-containing proteins SH-groupsof proteins Proteinscontaining transition metal

Proteinscontaining Dz~yl radical Oxygen Superoxide anion

1 Oxide

Examples/Effects Guanylate cyclase(+) Cyclooxygenase(t) Hemoglobin ( - ) GAPDH (-) Serum albumin NADH-ubiquinone oxidoreductase (-) NADH-succinate oxidoreductase(-) Cis-aconitase(-) Ribonucleotide reductase(-) Deamination Nitrite and nitrate production Peroxynitrite formation

Activation by nitric oxide: (+). Inhibition by nitric oxide: (-). GAPDH = glyceraldehyde-3-phosphate dehydrogenase; NADH = reduced form of nicotinamide adenine dinucleotide.

mining transition metal, such as the citric acid cycle enzyme aconitase, complex I (NADH-ubiquinone oxidoreductase) and complex II (succinate-ubiquinone oxidoreductase) of the mitochondrial electron transport chain, leading to inhibit mitochondrial respiration.37 Inhibition of ribonucleotide-reductase, a rate-limiting enzyme involved in DNA synthesis and repair, has also been shown.s7Under some conditions, nitric oxide nitrosylates free cysteine SH-groups of proteins and may, thus, inactivate proteins when cysteines are present in their active site.“’ Reaction of nitric oxide with the superoxide anion (O,.-) may yield peroxynitrite (ONOO-) , which is highly reactive and potentially a major cytotoxic agent mediating oxidative damage7,41.84 (Table 1). Finally, nitric oxide-mediated DNA damage leads to activation of poly(ADP-ribose) polymerase in the nucleus accompanied by NADt depletion and then cellular death 8%109

II. Localization of Different Nitric Oxide-Synthase Isoforms in the Eye A. CONSTITUTIVES

NOS (NO!%1 AND NOSIII)

Nitric oxide synthase enzymatic activity was demonstrated in crude extracts of the retina and in isolated rod outer segments (ROS) .26.46~102,107 Enzymatic properties indicate a relationship between the retinal enzyme and the brain constitutive isoform (type I). NADPHdiaphorase studies also indicate the presence of NOS in the retina.78”0” NOS-I isoform was clearly identified in some amacrine cells, in the inner nuclear layer and in photoreceptors of the retina from different speciesby immunohistochemistry46~8z~10R (Fig. 2). Recently, NOSI was identified in the retina of lower vertebrates5’ and cloned from the human retina.*’ The presence of NOS-III was not directly demonstrated by immunochemistry; however, the large NADPH-diaphorase staining observed in the vascular endothelium of anterior segment, choroid and retina”,“” could correspond to this isoform. Indeed, the presence of NOS-III mRNA has been found recently in retinal vascular endothelial cells” (Table 2). In addition, NOS can also be visualized outside blood vesselsin nerve endings present within anterior segment tissues.“” The ciliary muscle and outflow pathway (trabecular meshwork and Schlemms canal) of normal human eyes were found to be enriched in NADPH-diaphorase, the majority of which, by immunological analysis, consisted of NOS-1117’ (Table 2). B. INDUCIBLE

NOS (NOS-II)

In the retina, retinal Miiller glial cells can express the NOSH isoform after endotoxin and cvtokines

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Pigmented Enithelial Photorecentors

Horizontal

Cells

Amacrine Cells Inner Plexiform Layer (beaded Nerve Fibers) D&laced Amacrine Cells Vascular endothelium

$

VITREOUS

Fig. 2. Localization of differents NOS isoforms on the retina. By immunohistochemistry, NOS-I is identified in horizontal cells, in some amacrine cells and in the photoreceptors of the retina from different species. NOSII is produced, after endotoxin and cytokines stimulation, by retinal pigmented epithelial cells, retinal Miiller glial cells, and in retinal pericytes and capillary endothelial cells. NOS-III seems to be present only in the retinal and choroidal vascular endothelium.

tic cooperation between interferon y and lipopolysaccharid e and can be potentiated by tumor necrosis factor cq while costimulation with interferon y and interleukin-lb is essential for nitric ox-

stimulation.“’ Bovine,‘” human,‘” and murine5”$“’ retinal pigmented epithelial (RPE) cells also contain NOS-II (Fig. 2). In rat and bovine RI’E cells, NOSH mRNA and enzyme activity are induced by synergis-

TABLE

2

Localization and Potential Rolesof Nitric Oxide ProducedbyNOS-1 and NOS-III in theEye Tissue Cornea Conjunctiva Lens epithelium Vascular endothelium (ciliary and retinal vessels) Ciliav body, trabeculum Neural retina Amacrine cells Horizontal cells Photoreceptors

References

Function Unknown Vasodilatation, Vascular nermeabilitv Unknown Vasodilatation (blood pressure) Intraocular pressure Neurotransmission Neurotransmission Neurotransmission, Visual transduction

106 60 106 11,35,78,79,99,106 71,72,92,106 82,106 4,55,61,63 51,74,91,100

NITRIC

OXIDE

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IN THE EYE

localization of NOS by NADPHdiaphorase in the anterior part of the rat eye and demonstrated a staining in nerve fibers in the limbus, in the cornea (endothelium, epithelium and peripheral cornea) and in lens epithelium. Immunohistochemistry with an antiNO!% antibody did not recover the NADPHdiaphorase labeling in cornea1 epithelium and endothelium, or in lens epithelium, suggesting the presence of another NOS isoform or another oxidative enzyme possessing the NADPHdiaphorase properties. The role of nitric oxide in allergic conjunctivitis was studied in a pig model.“” Results indicated that nitric oxide was produced in the acute phase of allergic conjunctivitis, via NOS-III in endothelial cells, and mediated vasodilatation with increasing blood flow which led to increasing vascular permeability and subsequent edema formation.

ide production in human RPE cells. More recently, NOSII isoform has been detected in retinal pericytes and in capillary endothelial cells, in vitro after cytokines treatment” (Table 3). Cytokines and growth factors are key elements in the regulation of NOS-II induction.““‘7” We have demonstrated that fibroblast growth factors and transforming growth factor p have opposite actions on the regulation of the production of nitric oxide by these cells.” In bovine RF’E cells, fibroblast growth factor-l and fibroblast growth factor-2 inhibit the induction of NOS at the transcriptional level,“” while it does not inhibit NOS-II induction in rat or in human RF’E and rat retinal Miiller glial cells, despite the fact that these cells have fibroblast growth factor receptors.“x The opposite data were observed with transforming growth factor p (inhibition of nitric oxide production in human RPE cells and in rat RPE and retinal Mtiller glial cells), which frequently acts as an immunosuppressor signal, suggesting that the regulation of NOS activity in each cell depends on the status of the transduction signal activated by the cytokine network. Since several stimuli act simultaneously or sequentially upon cells, it is not surprising that cells and species-specific signals can be obtained. This is consistent with an efficient regulation of NOS expression at different levels, since cells must control tightly the formation of high amounts of nitric oxide.“”

B. TRABECULAR

CONJUNCTIVA,

AND LENS

No data were actually reported in the literature concerning the action of nitric oxide in cornea and lens. However, Yamamoto et al’“’ have studied the

TABLE Localization,

Conditions

Tissue or cells Iris/ciliary Neural

of Expression

and Putatiue

Role of NOS-II

Conditions/References

retina Light degeneration (i?~ vi~~o)~~~“’ CMV-retinitis (in viva) ” glial cells

Retinal pigmented

epithelial

Vessels (pericytes, endothelial

cells

cells)

Endotoxin and cytokines stimulation ( in vitro) :*” Endotoxin and cytokines stimulation (in z,&.o) X.Z.“7.‘L!I

Endotoxin

MUSCLE

3

body

Retinal Miiller

AND CILIARY

Several studies reported both ocular hypertension and hypotension induced by systemic administration of nitrovasodilatators.‘,“,‘4,4* In these studies, the observed effects on the intraocular pressure were seemingly due to their systemic vasodilatatory effects or to their action on regional blood flow. “‘.‘04.‘0R Some studies deal with the effects on intraocular pressure of guanylate cyclase activation by atria1 natriuretic peptides”*49.“‘,“7 or by nitrovasodilators. 19~6K~6y~7’1~q’ In the eye, multiple sites of action for the nitrovasodilators or nitric oxide donors include ciliary muscle, trabecular meshwork, and endothelial and vascular smooth muscle cells in the aqueous drainage system. The existence of these multiple sites of action is supported by anatomic studies,7’,“‘” which have demonstrated a distribution of NOS.1 restricted to ciliary processes and, in the outflow path-

III. The Role of Nitric Oxide in the Anterior Segment A. CORNEA,

MESHWORK

and cytokines stimulation

in the Eye Role Proinflammatory? Cytotoxicity Proinflammatory? Cytotoxicity Cytotoxicity Antiviral? Proinflammatory? Proinflammatory? Immune modulation? Antiphagocytic activity Antiproliferative Proinflammatory? Immune modulation? Vasodilatation (blood flow)

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ET AL

way, to a few nerve endings that appear to arise from inflammation of the anterior segment of the eye and the pterygopalatine and perhaps other parasympachorioretinal involvement. Cytokines are thought to thetic ganglia.lffi Furthermore, it has been shown contribute to the development of endotoxinthat ciliary muscle (especially its anterior longitudiinduced uveitis and possibly of human uveitis, which nal portion) and out.fIow pathway (trabecular meshis characterized by dilatation of iridal blood vessels, work, Schlemm’s canal, and collecting channels) of leakage of plasma proteins into aqueous humor, and normal human eyes were found to be enriched in infiltration of leukocytes into the uveal tract and NOS-III but not in NOS-I.‘l Other results reported aqueous humor. Recently, Parks et alB1 demonthe ability of nitrovasodilators and cGMP analogues strated that multiple intraperitoneal injections of to induce relaxation of trabecular meshwork and cilL-NAME, a well-known NOS inhibitor, reduced cliniiary muscle in ~itro’~~ and to alter outflow facility cal signs of uveitis in lipopolysaccharide-injected and decrease intraocular pressure in ~~~~~~~~~~~~~~~~~~ rats, suggesting that nitric oxide could participate to The involvement of the trabecular meshwork and the pathogenesis of endotoxin-induced uveitis as a Schlemm’s canal as a major site of outflow resisproinflammatory mediator. Furthermore, it has tance,*’ and the possible regulation of trabecular been shown that the hemodynamic and vascular permeshwork resistance by the ciliary muscle were also meability changes associated with endotoxinreported.21~43~86 These findings suggested that nitric induced uveitis in the rat are in part due to inoxide, acting at the ciliary muscle, outflow pathway, creased production of nitric oxide.‘s Recent or both, may be involved in the regulation of aqueworkss’*57demonstrate more directly the role of nious humor dynamics in humans. Recently, a detric oxide in endotoxin-induced uveitis. Expericrease of NADPHdiaphorase staining and distribuments have been conducted which demonstrated tion in trabecular meshwork and Schlemm’s canal high levels of nitrite in the aqueous humor and vitrehas been described in patients with primary openous of rats at the time of maximal ocular inflammaangle glaucoma, with a marked reduction of antetion following injection with lipopolysaccharide,“’ rior longitudinal ciliary muscle fibers that terminate and the presence of a calcium independent NOS acnear the trabecular meshwork.” The question we activity in the anterior segment during endotoxin-intually cannot answer is whether this defect is associduced uveitis. 57Furthermore, reverse transcriptaseated with the cause of primary open-angle glaucoma, PCR assaysshow an increased expression of NOSII or whether it is a result of the disease.Furthermore, mRNA in the iris-ciliary body and in the retina of although this defect appears simultaneously with rats with endotoxin-induced uveitis.3’ In situ hybridglaucoma, we could not exclude that it has no direct ization revealed that epithelial cells of the iris-ciliary relationship with the pathology. In glaucoma, inbody and cells infiltrating (macrophages, polymorcreased intraocular pressure could be caused by rephonuclear leukocytes) the anterior segment and in duction or absence of longitudinal ciliary muscle fithe retina are the major source of nitric oxide.4’ Morever, a significant correlation was obtained bebers. The contractions of these muscle fibers lower tween the nitrite level in aqueous humor and cliniintraocular pressure through a decrease in outflow resistance.5’43’Rh cal signs of endotoxin-induced uveitis. A similar correlation was found between nitrite detected in the These findings raise the possibility of potential vitreous body and endotoxin-induced uveitis.“’ The therapeutic uses of the nitrovasodilators. Because efficacy of treatment with NOS inhibitors (No-nitrothese agents lack vasoconstrictive effects, unlike curL-arginine methyl ester, aminoguanidine) in rat3’,” rent ocular hypotensives, and have the potential of or in rabbi?’ in endotoxin-induced uveitis suggests increasing retinal blood flo~,~~ they might also exert that treatments that reduce nitric oxide production beneficial effects on injured retinal cells in glauor nitric oxide action would be beneficial and would coma. 53These compounds could be particularly useprovide new prospects for treatment of human uveiful in glaucoma patients in whom a systemic tentis. It would be interesting to test whether topical addency for vasospasmhas been identified.17.36 ministration of NG-nitro-L-arginine methyl ester or a IV. Nitric Oxide and Uveitis more selective inhibitor of NOS-II to the eye is able to locally inhibit NOS activity without adverse effects. Endotoxin-induced uveitis serves as a model for certain types of human ocular inflammation, collectively termed uveitis, that appear in Reiter’s synV. Nitric Oxide in the Retina drome, dysentery syndromes, Crohn’s disease,ulcerA. NITRIC OXIDE IN SYNAPTIC TRANSMISSION ative colitis, sarcoidosis, and Behcet’s disease.‘* A 1. Neurotransmission single intravitreal injection of lipopolysaccharide in NOS has been found to be located both pre- and rabbits, *’ like a systemi c injection of lipopolysacchapostsynaptically, and recent physiological studies ride in rodents,““,8838g induces acute uveitis with acute

77

NITRIC OXIDE IN THE EYE have implicated its involvement in a variety of phenomena from longterm potentiation (a form of memory) to excitotoxicity.g In the retina, different experiments suggest that nitric oxide could serve as a neuromodulator of synaptic transmission, via a modification of electrical coupling in horizontal cells,‘j” activation of cGMP-gated conductance in bipolar cells”” and modulation of a cGMP-gated conductance in ganglion cells.’ Several studies suggest that electrical coupling is thought to influence the lateral spread of light information in the retina by altering the receptive field size of the horizontal cells~4,55&‘,“:4 2. Phototransduction Nitric oxide could modulate visual transduction because it has been shown that it can modulate conductance of some photoreceptor ion channels.5’ These properties of nitric oxide seem very specific, since the nitric oxide generating compound S-nitrocysteine increased calcium channel current and a voltage-independent conductance, but had no effect on nonspecific cation current. Furthermore, some electrophysiological studies, performed on isolated rod outer segments show that nitric oxide alters outer segments dark voltage and light response74.g1,1”” and induces ADP-ribosylation of photoreceptor proteins, including transducin.‘s.*“.“” B. POTENTIAL ROLE OF NITRIC IN RETINAL DISEASES

OXIDE

During the last two years, the possibility that nitric oxide could be one of the mediators involved in the pathogenesis of retinal pathologies or degeneration, has been investigated. 1. Nitric Oxide and Retinitis Recently, we demonstrated that NOS-II is expressed in vivo in human retina asa result of viral infection.‘” Indeed, NOSII has been detected in cytomegalovirus (CMV)-infected retina from AIDS patients by immunohistochemistty and was localized to (XV-infected glial cells (astrocytes and retinal Miiller glial cells). High levels of interferon y were found in the eyes of AIDS patients, suggesting that this cytokine could contribute to NOSII induction. The role of nitric oxide in viral infections of the retina could be beneficial via its antiviral effects,50*7” but also detrimental through its potential to damage tissue. More recently, NOSII induction was demonstrated in the retina of rodents presenting endotoxin-induced uveitis (see IV. Nitric Oxide and Uveitis) . 2. Nitric Oxide and Retinal Degeneration The role of high levels of nitric oxide produced by NOS-II in the retina is poorly understood. Benefi-

cial antimicrobial, antitumoral and antiviral effects of nitric oxide could be considered. However, we could speculate that it would perturbate the different physiological processesof the retina. We reported that exogenous nitric oxide, via nitric oxide donor or endogenous nitric oxide, in lipopolysaccharide/interferonstimulated cells could decrease RPE cell phagocytic activity in vitro, as evaluated by the rod outer segments (ROS) ingestion in a radio-immunoassay.” Furthermore, addition of the potent inhibitor of NOS, N’;-monomethyl-L-arginine (L-NMMA) , which inhibits nitrite releaseby lipopolysaccharide/interferon-stimulated RPE cells, restored normal phagocytosis of ROS membranes, demonstrating that endogenous nitric oxide is the compound involved in lipopolysaccharide/interferon- decreased RPE phagocytic activity. We suggest that the production of large amounts of nitric oxide in the retina could be deleterious by perturbing ROS membrane phagocytosis by RPE cells. This inhibition could lead to the accumulation of ROS debris between photoreceptors and retinal pigmented epithelium and ultimately result in photoreceptor degeneration. It has been proposed that light-induced, oxygenfree radicals may be mediators of retinal photic injury, since several reports have demonstrated the protective effect of free radical scavengers in the light damage model.‘“~” More recently, the involvement of nitric oxide has been suggested in lightinduced photoreceptor degeneration. Indeed, by measuring the photoreceptor nuclear layer thickness, we found that intraperitoneal injection of the inhibitor of NOS, N’;-nitro-L-arginine methyl ester (L-NAME), into albino Fisher rats maintained in constant light for seven days, partially protects against the degeneration of photoreceptors and acts to maintain their morphological organization with a more pronounced effect observed in the superior hemisphere. ‘s Reverse transcrip tase-PCR experiments, which allow the detection of small amounts of mRNA, revealed that constant illumination induced NOS-II in the retina in uivo and that this induction correlated with photoreceptor degeneration.“’ The cellular type or types that are the primaly source of nitric oxide in the retina, namely resident cell types (RPE or retinal Muller glial cells) or infiltrating cells (macrophages), are not known. The involvement of NOSI and NOS-III has not been investigated, but their roles should not be excluded. A number of different pathways could be involved in the nitric oxide-induced light degenerative process: nitric oxide might act by eliciting a cGMP increase in the photoreceptor cells leading to excess calcium influx.51.‘4 Nitric oxide could ADP-ribosylate certain photoreceptor proteins and modulate their activity. “.“(’ Nitric oxide could also act as a free radical

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capable of combining with oxygen derivatives, resulting in the production of the peroxynitrite anion, ONOO-, which is highly cytotoxic.‘,” Alternatively, it is possible that an excess of nitric oxide might impair the capacity of RPE cells to ingest photoreceptor outer segments, as previously reported* and lead to photoreceptor destruction. 3. Nitric Oxide and Other Retinal Diseases Nitric oxide was able to inhibit proliferation of RPE cells in vitro. ” The nitric oxide was obtained either by the addition of a nitric oxide donor or by endogenous nitric oxide produced upon stimulation with lipopolysaccharide and cytokines. It is thus possible that in retinal pathologies where RPE cells proliferate as in proliferative vitreoretinal diseases,56 production of nitric oxide may participate in the control of cell growth. It will be of great interest to determine the relationship between nitric oxide levels in the vitreous of patients suffering from proliferative vitreoretinopathy (PVR) as a function of the evolution of the PVR pathology (inflammation, cell proliferation). A beneficial role of nitric oxide for the survival of injured retinal ganglion cells after axotomy has been recently reported.40 Indeed, injection of NOS inhibitor (L-NAME) following axotomy increased loss of retinal ganglion cells over two weeks, suggesting that the physiological release of nitric oxide by constitutive NOS isoforms I and III in the retina will improve the survival of retinal ganglion cells after axotomy. Finally, the production of nitric oxide by vascular endothelium36 and certain nerve endings could play a vasodilator role in ophthalmic artery and in retinal arterioles.35’gg This nitric oxide release, which controls arteriolar tone, could be measured directly at the retinal surface with a nitric oxide microprobe inserted in the vitreous of a miniature pig.‘” Recent data demonstrates that nitric oxide is involved in a major way in the mechanism of ischemic damage and may promote neuronal cell death via free radical production and N-methyl-D-aspartate-receptormediated toxicity.143103 Because of its vasodilatatory property, nitric oxide will also improve blood flow during or immediately after ischemia. Thus, paradoxically, nitric oxide production, initiated immediately after the onset of ischemia, can significantly attenuate the volume of damaged tissue. Some groups have studied the effect of NOS inhibition in retinal ischemia. One group showed that NOS inhibitors protect rat retina against ischemic injury.24 However, another group found that retinal functional recovery after ischemia ended, as assessed by the electroretinogram, was not different between saline or L-NAME-treated cats.” These authors demonstrated that nitric oxide is also capable of increasing blood

BECQUET ET AL flow during or immediately after ischemia in the retina but not in the choroid.” It seems that differences between these tissues account for lack of reaction in the choroid. In fact, the precise contribution of nitric oxide to the pathogenesis of retinal ischemic damage remains controversial. Further investigations are necessary to assess more completely the role of nitric oxide in retinal ischemia, and an important feature is to distinguish between the respective role of the different NOS isoforms and the possibility of regulating specifically their expression during ischemia and/or reperfusion phase. The retina is damaged during the ischemic episode, but, paradoxically, it also may be damaged after blood flow restoration, a phenomenom which implicates oxygen free radical formation.23’76 The presence of superoxide anion is an important feature of the retina, since it could combine with nitric oxide to give rise to peroxynitrite, a very toxic compound, as suggested above.” Thus, it is probably the combination of these two different free radicals during reperfusion which is toxic to the cells.

VI. Conclusion and Therapeutic Directions Initial research on nitric oxide in the eye has focused attention on the importance of this regulatory pathway in vascular, immunological, and retinal physiology. Further research will be needed to understand the contribution of the nitric oxide pathway to the etiology of eye diseases and, in particular, the regulation of inducible NOS isoform, which may contribute to a number of pathophysiological states. Among the first therapeutic uses suggested for NOS inhibitors have been sepsis and septic shock associated with deep hypotension, consecutively to NOS-II induction.643101 The enhanced formation of nitric oxide could disrupt the functional retinal integrity, could lead to retinal inflammation and then could participate to retinal degeneration. In the case of endotoxininduced uveitis and probably of other ocular inflammatory pathologies where NOM1 is induced, it is possible that a topical administration of NOS inhibitors to the eye, allowing local inhibition of NOS activity, might improve symptoms of these diseases. NOS inhibition could also provide a new therapeutic approach to retinal degeneration since nitric oxide seems to be an important mediator of light induced retinotoxicity.31 In the central nervous system, a modulation of nitric oxide release seems to emerge in therapy for ischemia,‘43’03 and the same approach could be also envisaged for retinal ischemia. However, if nitric oxide could play a therapeutic role in retinal ischemia, it will be necessary to determine the relative contribution of constitutive NOS (NOM and -1111) and in-

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ducible NOS (NOS-II) during the &hernia process.Because large release of nitric oxide via NOS-II could be responsible for tissue damage during ischemia, it would be profitable to inhibit specifically this isoform, without inhibiting the vascular endothelial isoform NOSIII, which could improve blood flow during or immediately after ischemia.14 Presently available NOS inhibitors are neither particularly potent nor selective, but the availability of such agents will help to elucidate the various postulated roles of NOS in regulatory systems. Then, specific NOS-II inhibitors will have the advantage of specifically inhibiting NOM1 isoform expressed in pathological states and will not perturb constitutive NOS-I and NOM11 activities, which seemsessential in the regulation of ocular blood flow, intraocular pressure, or retinal physiology. Current evidence indicates that nitric oxide is involved in maintaining basalvascular tone in uveal, retinal and choroidal circulations. In this context nitric oxide donors could potentially have positive actions in pathologies, such as glaucomatous optic neuropathy or retinal vascular occlusion, where optic nerve, retinal or ophthalmic artery blood flow should be increase. However, a better understanding of nitric oxide transduction pathway may lead to more efficient nitrovasodilators or agents which might mimic or modulate the role of nitric oxide in vasodilatation. Other experiments reported that nitric oxide was able to decrease intraocular pressure in normal During glaucoma, the application of a noneYeS.6g~g? toxic nitric oxidegenerating compound could restore a normal intraocular pressure, and then represent a new class of antiglaucomatous treatment. However, many experiments must be conducted to demonstrate the lack of toxicity of such nitric oxidegenerating compounds in the anterior segment of the eye. Finally, the potential use of NOS inhibitors (ocular inflammatory diseases,retinal degeneration) or nontoxic nitric oxide donors (glaucoma) reflects, for the moment, hypothetical or experimental perspectives. A better understanding of this nitric oxide pathway will be the key for the development of new approaches to the management and treatment of various eye diseases.

5.

6.

7.

8.

9. 10

11.

12.

13. 14.

15.

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Outline I

Characteristics of nitric oxide A. Biosynthesis of nitric oxide B. Chemical reactivity and molecular targets of nitric oxide II. Localization of different NOS isoforms in the eye A. Constitutives NOS (NOM and NOS-III) B. Inducible NOS (NOS-II) III. The role of nitric oxide in the anterior segment A. Cornea, conjunctiva, and lens B. Trabecular meshwork and ciliary muscle

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IV. Nitric oxide and uveitis V. Nitric oxide in the retina A. Nitric oxide in synaptic transmission 1. Neurotransmission 2. Phototransduction B. Potential role of nitric oxide in retinal diseases 1. Nitric oxide and retinitis 2. Nitric oxide and retinal degeneration 3. Nitric oxide and other retinal diseases VI. Conclusion and therapeutic directions

We are grateful to Dr. G. Caputo for critical reading manuscript. Reprint address: F. Becquet, MD, PhD, INSERM U450, Wilhem. 75 016 Paris, France.

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