Hydrogen Sulfide Protects the Retina from Light-inducedDegeneration by the Modulation of Ca2� Influx*

Received for publication, August 26, 2011, and in revised form, September 19, 2011 Published, JBC Papers in Press, September 20, 2011, DOI 10.1074/jbc.M111.298208

Yoshinori Mikami‡, Norihiro Shibuya‡, Yuka Kimura‡, Noriyuki Nagahara§, Masahiro Yamada¶, and Hideo Kimura‡1

From the ‡Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology andPsychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan, the §Department of Environmental Medicine, Nippon MedicalSchool, Bunkyo-ku, Tokyo 113-8602, Japan, and the ¶Laboratory for Neuroinformatics, RIKEN Brain Science Institute, Wako,Saitama 351-0198, Japan

Background: Hydrogen sulfide (H2S) has been recognized as a signaling molecule as well as a cytoprotectant.Results:Ca2� regulates the 3-mercaptopyruvate sulfurtransferase/cysteine aminotransferase pathway to produce H2S produc-tion. H2S, in turn, regulates Ca2� influx and protects retinal neurons from light-induced degeneration.Conclusion: H2S regulates Ca2� levels and protects retinal neurons.Significance: It provides a possible role of H2S and its therapeutic application in the retina.

Hydrogen sulfide (H2S) has recently been recognized as a sig-naling molecule as well as a cytoprotectant. Cystathionine�-synthase (CBS) and cystathionine �-lyase (CSE) are well-known as H2S-producing enzymes. We recently demonstratedthat 3-mercaptopyruvate sulfurtransferase (3MST) along withcysteine aminotransferase (CAT) produces H2S in the brain andin vascular endothelium. However, the cellular distribution andregulation of these enzymes are not well understood. Here weshow that 3MST and CAT are localized to retinal neurons andthat the production of H2S is regulated by Ca2�; H2S, in turn,regulates Ca2� influx into photoreceptor cells by activating vac-uolar type H�-ATPase (V-ATPase).We also show that H2S pro-tects retinal neurons from light-induced degeneration. Theexcessive levels of light exposure deteriorated photoreceptorcells and increased the number of TUNEL- and 8-hydroxy-2�-deoxyguanosine (8-OHdG)-positive cells. Degeneration wasgreatly suppressed in the retina of mice administered withNaHS, a donor of H2S. The present study provides a new insightinto the regulation ofH2S production and themodulation of theretinal transmission byH2S. It also shows a cytoprotective effectof H2S on retinal neurons and provides a basis for the therapeu-tic target for retinal degeneration.

Hydrogen sulfide (H2S) is synthesized by three enzymes: cys-tathionine �-synthase (CBS),2 cystathionine �-lyase (CSE), and

3-mercaptopyruvate sulfurtransferase (3MST) along with cys-teine aminotransferase (CAT) (1–6). 3MSTproducesH2S from3-mercaptopyruvate (3MP), which is produced from cysteineand �-ketoglutarate (�-KG) by CAT that is identical withaspartate aminotransferase (4, 7, 8). In the central nervous sys-tem, CBS is mainly localized to astrocytes, and 3MST to neu-rons (4, 9, 10). H2S facilitates the induction of hippocampallong-term potentiation by enhancing the activity of NMDAreceptors and induces Ca2� waves in astrocytes (2, 11, 12). Italso relaxes smooth muscle by activating KATP channels, regu-lates insulin release and induces angiogenesis (3, 13–16).In addition to its role as a signalingmolecule, H2S has a cyto-

protective effect. H2S protects neurons from oxidative stress byenhancing the activity of �-glutamylcysteine synthetase(�-GCS) as well as the transport of cysteine and cystine, leadingto reinstating the levels of GSH decreased by oxidative insults(17, 18). H2S also protects cardiac muscle from ischemia-rep-erfusion injury (19). H2S facilitates the nuclear localization of atranscription factor, nuclear factor erythroid 2-related factor 2(Nrf2), which increases the expression of antioxidants such asthioredoxin and heme-oxygenase1 (20). H2S produced by3MST along with CAT can directly react with several cytotoxicoxidant species in mitochondria to protect cells (18, 21).Calcium ion (Ca2�) acts as a second messenger involved in a

broad spectrum of intracellular signaling pathways. A numberof enzymes are regulated by Ca2�. NOS and heme oxygenase-2are regulated by Ca2�/calmodulin (22, 23). There are severalenzymes that are directly regulated by Ca2� without beingmediated by Ca2�-binding proteins. For examples, three intra-mitochondrial citrate cycle dehydrogenases; pyruvate dehydro-genase,NAD-isocitrate dehydrogenase, and oxoglutarate dehy-drogenase were activated by Ca2� (24). Recently, wedemonstrated H2S production by 3MST depends on thiore-doxin and dihydrolipoic acid (DHLA) (25). However, the regu-lation of the activity of 3MST is not well understood.

* This work was supported by a grant from the National Institute of Neurosci-ence and by KAKENHI (23659089) from Grant-in-Aid for ChallengingExploratory Research (to H. K.), by KAKENHI (23790316) from Grant-in-Aidfor Young Scientists (B) (to Y. M.), by KAKENHI (23700434) from Grant-in-Aid for Young Scientists (B) and Health Labor Sciences Research Grant fromthe Ministry of Health Labor and Welfare (to N. S.), and by KAKENHI(22590258) from Grant-in-Aid for Scientific Research (C) (to Y. K.).

1 To whom correspondence should be addressed: 4-1-1 Ogawahigashi, Kod-aira, Tokyo 187-8502, Japan. Tel.: 81-42-346-1725; Fax: 81-42-346-1755;E-mail: kimura@ncnp.go.jp.

2 The abbreviations used are: CBS, cystathionine �-synthase; 3MP, 3-mercap-topyruvate; 3MST, 3-mercaptopyruvate sulfurtransferase; 8-OHdG, 8-hy-droxy-2�-deoxyguanosine; Baf A1, bafilomycin A1; BBS, bicarbonate buffersaline; CAT, cysteine aminotransferase; CSE, cystathionine �-lyase; DHLA,dihydrolipoic acid; GCL, ganglion cell layer; INL, inner nuclear layer; IPL,

inner plexiform layer; �-KG, �-ketoglutarate; NEM, N-ethylmaleimide; ONL,outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; PLP,pyridoxal 5�-phosphate; ROS, reactive oxygen species; Trx, thioredoxin;V-ATPase, vacuolar type H�-ATPase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 45, pp. 39379 –39386, November 11, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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The present study shows that the H2S production by 3MSTand CAT is regulated by intracellular Ca2�. H2S, in turn, sup-presses Ca2� channels by activating vacuolar type H�-ATPase(V-ATPase). Under physiological conditions, H2S may main-tain intracellular Ca2� in low levels. The regulation of Ca2� byH2S may be failed by the excessive levels of light, and the pho-toreceptor cell degeneration occurs. Even under such condi-tions the administration of sodium hydrosulfide (NaHS), adonor of H2S, suppresses photoreceptor degeneration. Theseobservations suggest that H2S protects photoreceptor cellsfrom the insult caused by excessive levels of light.

EXPERIMENTAL PROCEDURES

Chemicals—Bafilomycin A1, calcium chloride, L-cysteinehydrochloride monohydrate, DTT, eosin Y,N-ethylmaleimide,magnesium sulfate, Mayer’s hematoxylin solution, mercapto-pyruvate sodium salt, �-ketoglutarate, potassium chloride,potassium dihydrogen phosphate, sodium chloride, sodiumhydrogen carbonate, sodium sulfide, and sucrose were pur-chased from Wako Pure Chemicals Industries (Osaka, Japan).Bicuculline methiodide, CremophorEL, diltiazem hydrochlo-ride, EGTA, glucose, nifedipine, picrotoxin, pyridoxal 5�-phos-phate monohydrate, sodium hydrosulfide, and Tween 20 werepurchased from Sigma-Aldrich (St. Louis, MO). Calmodulinwas purchased fromMerckCalbiochem (Darmstadt, Germany)and W-7 from BIOMOL International (Plymouth Meeting,PA).Determination of H2S Producing Activity—All animal proce-

dures were approved by the National Institute of NeuroscienceAnimal Care and Use Committee. Homogenates of the mouseretina were prepared in the ice-cold isolation buffer containing50 mM potassium phosphate (pH7.4), 1 mM DTT, the proteaseinhibitor mixture complete EDTA-free (Roche Diagnostics,Mannheim,Germany) and 10mMEGTA, and sonicated for 10 susing a sonicator (Branson Model 450; Branson Ultrasonics,Danbury, CT). Protein concentrationswere determined by Bio-Rad Dc Protein Assay (Bio-Rad Laboratories, Hercules, CA)according to the manufacturer’s instructions.The enzyme reaction was performed as described (4) with

modifications. The substrate was added to 0.1 ml of homoge-nates or cell lysates in a 15 ml centrifuge tube and incubated at37 °C for 30 min. After adding 0.2 ml of 1 M sodium citratebuffer, pH 6.0, the mixtures were incubated at 37 °C for 10 minwith shaking on a rotary shaker to facilitate a release of H2S gasfrom the aqueous phase. Two ml of approximate 14.5 ml ofhead-space gas was applied to a gas chromatograph (GC-2014;Shimadzu, Kyoto, Japan) equipped with a flame photometricdetector and a data processor C-R8AChomatopac (Shimadzu).The concentrations of H2S were calculated using a standardcurve of 0 to 5 nM of Na2S, as a source of H2S.Constructs and Transient Transfection of HEK 293-F Cells—

The constructs of 3MST and CAT expression plasmids weredescribed previously (4). Transient transfection of HEK 293-Fcells in suspension cultures was performed using a FreeStyle293 Expression System (Invitrogen Life Technologies Corp.,Carlsbad, CA). HEK 293-F cells efficiently express externallyapplied expression plasmids and grow in higher densities insuspension cultures than regular plating. For transfection, 10

�g of expressionplasmidsweremixedwith 15�l of transfectionreagent 293 fectin (Invitrogen) and then added to 1 � 107 cellsin 100ml Erlenmeyer flasks with 10ml of FreeStyle 293 expres-sion medium. Cells were incubated with shaking at 125 rpm ona rotary shaker at 37 °C in a humid atmosphere with 10% (v/v)CO2. Transfection efficiencywas�90%.Cells were harvested at48 h post-transfection. The HEK 293-F cells were precipitatedby centrifugation at 1000 � g for 5 min. After washing withice-cold PBS, cell pellets were resuspended in ice-cold buffer Aand sonicated.Immunohistochemistry—Mouse eyecups were fixed over-

night at 4 °C with 4% (w/v) paraformaldehyde in PBS. Afterrinsing in PBS, tissues were cryoprotected in 5, 15, and 30%(w/v) sucrose/PBS in order and embedded in Tissue-TekO.C.T. compound (Sakura Finetechnical, Tokyo, Japan) in analuminum boat and frozen in liquid nitrogen. Retinal sectionswith 12-�m thickness were cut by a cryostat (CM1900, LeicaInstruments, Nussloch, Germany) at �20 °C and mounted onMAS-coated slides (Matsunami, Osaka, Japan) and dried. Thesections were incubated with 10% (v/v) normal goat serum(Nichirei, Tokyo, Japan) or blocking solution (Roche Diagnos-tics) at room temperature for 1 h. For double immunofluores-cence staining, sections were incubated overnight at 4 °C withantibodies against rabbit anti-3MST polyclonal antibody(1:3000) (26), sheep anti-aspartate aminotransferase (AST;cytosolic CAT) antibody (1:3000; Rockland, Gilbertsville, PA),sheep anti-glutamic-oxaloacetic transaminase 2 (GOT2; mito-chondrial CAT) antibody (1:1000; Lifespan biosciences, Seattle,WA), mouse anti-calbindin-D antibody (1:1000; clone CB-955,Sigma), rabbit anti-CSE antibody (1:3000) (27), rabbit anti-CBSantibody (1:3000) (9), or mouse anti-8-hydroxy-2�-deox-yguanosine (8-OHdG) antibody (5 �g/ml, Japan Institute forthe Control of Aging, Nikken SEIL, Shizuoka, Japan). Theimmunoreaction was visualized with secondary subclass-spe-cific Alexa Fluor 488-, Alexa Fluor 555-conjugated (Invitrogen)or Cy3-conjugated (Jackson ImmunoResearch laboratories,West Grove, PA) antiserum at a dilution of 1:200. For nuclearstaining, sections were incubated with 2 �g/ml Hoechst 33342(Invitrogen) in PBS for 2 min at room temperature. The speci-mens were observed under a laser confocal microscope (TCSSP2; Leica Microsystems, Wetzlar, Germany) mounted on aLeica Microsystems inverted microscope (DMIRE2) using theoil-immersion objective (63�; 1.32 NA; Leica Microsystems).Preparation of Mouse Retinal Slices and Ca2� Imaging—The

mice were kept in a room maintained under a 12 h/12 h lightcycle.Mice were dark-adapted for 2 h before experiments. Sub-sequent manipulations were performed under dim red light.The mice were anesthetized and killed by cervical dislocation.The eyes were enucleated and immediately put in ice-coldbicarbonate-buffered saline (BBS), composed of: 120mMNaCl,22.6mMNaHCO3, 3mMKCl, 0.5mMKH2PO4, 2mMCaCl2, 0.5mM MgSO4, and 6 mM glucose. The BBS solution (pH 7.4) wascontinuously bubbled with 95% (v/v) O2 and 5% (v/v) CO2.After enucleation, the anterior segment of the eye including thelens was removed. The resulting eyecup was detached from thepigmented epithelium, and a section of retinawas placed vitrealside down on a piece of filter paper (13 mm in diameter, TypeHAWP, 0.45 �m pores, Millipore, Billerica, MA). After adher-

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ing to the filter paper, retinal slices with 200–250�m thicknesswere manually cut with a razor blade.For Ca2� imaging, retinal slices were loaded with a cell-per-

meant Ca2� indicator, Calcium Green-1 AM (Invitrogen), byincubating slices for 50 min in the dark with 1 �M CalciumGreen-1 AM and 0.01% (v/v) CremophorEL in BBS solution.The slices were positioned in the glass-bottom dish (Matsu-nami) with high-pure white vaseline (Wako) for viewing theretinal layers. The dish was mounted on an upright fixed stagemicroscope (Leica DMLFS, Leica Microsystems). The imageswere acquired using a xenon lamp (Osram, Augsburg, Ger-many), a water immersion objective (40X; 0.80 NA; LeicaMicrosystems) and a CCD camera (C4742–95-12ER;Hamamatsu Photonics, Shizuoka, Japan). Excitation and emis-sion were controlled by LEPMAC5000 filter wheel and a shut-ter controller (Ludl electronic products, Hawthorne, NY).Frame duration ranged from 20–35 ms and each image wasacquired at 5-s intervals. Images were acquired and analyzedusing Aquacosmos software Ver. 2.0 (Hamamatsu Photonics).Light Damage—Male ICR mice (Japan Clea), aged 8–10

weeks were kept under controlled lighting conditions (12h-light/12h-dark). After dark adaptation for 24 h, NaHS (0.4375�mol/kg) or vehicle (PBS) was administered intraperitoneallytomice 15min before exposure to light. Mice were dilated with5% (w/v) phenylephrine eye drops (Kowa, Tokyo, Japan) 5 minbefore exposure to light. Nonanesthetized mice were exposedto while fluorescent light (1220 lm) for 2 h in cage (375 cm2)with reflective interior. After light exposure, animals remainedin darkness until they are analyzed.For analysis with a light microscopy, the eyes were enucle-

ated under dim red illumination, fixed with 4% (w/v) parafor-maldehyde in PBS at 4 °C overnight and immersed in 5, 15, and30% (w/v) sucrose in PBS in order at 4 °C. The eyes were thensubmerged into an O.C.T. compound and frozen. The retinalsections with 12-�m thickness were cut by a cryostat at�20 °Cand stored at �80 °C until staining. The sections were stainedwith Mayer’s hematoxylin and eosin for histological analysis.To detect the retinal cell death, TUNEL staining was per-formed. The sections were washed three times in PBS and incu-bated in PBS containing 2 �g/ml proteinase K (Merck) at 37 °Cfor 10 min. The sections were washed three times in PBS andincubated in 0.3% (w/v) hydrogen peroxide with methanol atroom temperature for 5 min to block endogenous peroxidaseactivity. The sectionswerewashed and incubatedwith terminaldeoxyribonucleotidyl transferase (TdT; Invitrogen) with bio-tin-labeled dCTP (Invitrogen) at 37 °C for 1 h. The biotin-la-beled dCTP was detected colorimetrically using peroxidase-conjugated streptavidin (Nichirei) and its chromogenicsubstrate 3, 3�-diaminobenzidine (Dojindo, Kumamoto, Japan).Sections were imaged by an epifluorescencemicroscopy (Axio-phot, Carl Zeiss, Germany) using a Plan-NEOFLUAR 40�objective (Carl Zeiss). Three light-microscope images werephotographed from each eye sample, and the number ofTUNEL-positive cells in the outer nuclear layer was averagedfor five eye samples.Statistical Analysis—All statistical analyses of the data were

performed using Microsoft Excel 2004 for Mac (Microsoft,Redmond, WA) with the add-in software Statcel2 (OMS,

Saitama, Japan). Differences between two groupswere analyzedwith Student’s t test. Differences between three ormore groupswere analyzed with one-way analysis of variance (ANOVA).

FIGURE 1. Localization of 3MST and CAT in the retina. A, schematic repre-sentation of the production of H2S by 3MST and CAT. Cys, cysteine; �-KG,�-ketoglutarate; 3MP, 3-mercaptopyruvate; Trx (ox), oxidized form of thiore-doxin; Trx (red), reduced form of thioredoxin; DHLA, dihydrolipoic acid; �-LA,�-lipoic acid. B–I, 3MST (B, F) co-localizes with cytosolic CAT (cCAT, C) andmitochondrial CAT (mCAT, G). 3MST and cCAT (D) or mCAT (H) are merged.J–Q, 3MST is localized to horizontal cells. 3MST (J, N) colocalizes with calbindin,a specific marker of horizontal cells (K, O). 3MST and calbindin were merged (L, P).The square area in L is magnified and shown in N–Q. R–U, neither CBS (R) nor CSE(T) is found in the retina. Nuclei were stained by Hoechst 33342 (E, I, M, Q, S, U,respectively). B–U, scale bars, 20 �m. OS, outer segment; IS, inner segment; ONL,outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, innerplexiform layer; GCL, ganglion cell layer.

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Post hoc multiple comparisons were made using the Bonfer-roni/Dunn test.

RESULTS

3MST and CAT Are Localized to the Retinal Neurons—Be-cause H2S-producing enzymes 3MST and CAT are localized toneurons in the brain (4), we examined the localization of bothenzymes in the retina by immunohistochemistry (Fig. 1A). Both3MST and CATs were localized to the inner plexiform layer,the outer plexiform layer, the inner nuclear layer, the outernuclear layer and the outer segments of photoreceptors (Fig. 1,B--I). Especially 3MST co-localized with calbindin, a specificmarker for horizontal cells (Fig. 1, J–Q). Neither CBS nor CSEwas found in the retina (Fig. 1, R–U).The Production of H2S by 3MST and CAT Is Regulated by

Ca2�—Because both 3MST and CAT are localized in the ret-ina, it is possible that the retina can produce H2S. The produc-tion of H2S by lysates of the retina was examined. Lysates pro-duced H2S in the presence of cysteine, �-KG and pyridoxal5�-phosphate (PLP) (Fig. 2A). In the absence of �-KG, which isrequired for CAT but not for CBS nor CSE, lysates producedlittle H2S (Fig. 2A). These observations suggest that H2S is pro-duced by 3MST along with CAT in the retina.

Because Ca2� regulates the activity of many enzymes, weinvestigated the effect ofCa2�on the activity of 3MSTandCATby measuring the amount of H2S produced by retinal lysates.H2S production was the maximum in the absence of Ca2� andsuppressed by Ca2� in a dose-dependentmanner (Fig. 2B). Theproduction of H2S was completely suppressed at 2.9 �M Ca2�.Since the intracellular concentrations of Ca2� change from�600 nM in darkness to less than 10 nM during illumination inmouse retinal photoreceptor cells (28), H2S is likely to be pro-duced by 3MST and CAT when photoreceptor cells areexposed to light.Because of its role in Ca2�-dependent regulation, the

involvement of calmodulin in the Ca2� regulation of H2S pro-duction was examined. Neither calmodulin nor a calmodulinspecific inhibitor, W-7, showed any effect on the production ofH2S in cell lysates (Fig. 2C). These observations suggest thatcalmodulin is not involved in the regulation of H2S-producingactivity of 3MST and CAT.To determine which enzyme, 3MST or CAT, is regulated by

Ca2�, H2S production from lysates of HEK 293-F cells overex-pressing 3MST and CAT was examined. To evaluate Ca2�

dependence of 3MST, the amount of H2S was measured using3MP as a substrate in the absence of CAT. The production of

FIGURE 2. H2S production by 3MST along with CAT is regulated by Ca2�. A, H2S production requires cysteine and �-KG in the retina. Lysates of the retinawere mixed with 2 mM cysteine, 0.5 mM �-KG, or 0.05 mM PLP and 17 nM Ca2�. **, p � 0.01 versus the mixture of cysteine, �-KG and PLP. B, H2S production fromcysteine and �-KG depends on Ca2�. Lysates of the retina produced H2S in the presence of cysteine, �-KG and Ca2� with the concentrations indicated. *, p �0.05 versus Ca2� free control. C, calmodulin is not involved in the regulation of H2S production from cysteine and �-KG. The production of H2S from cysteine and�-KG by retinal lysates was not changed in the presence of 1 �M calmodulin or 100 �M W-7, a calmodulin inhibitor. (�): control. NS, not statistically significantbetween indicated groups. D, H2S production by 3MST from 3-mercaptopyruvate (3MP) does not depend on Ca2�. The lysates of HEK 293-F cells expressing3MST were incubated with 0.01 mM 3MP. E and F, H2S production by 3MST and CATs depends on Ca2�. The lysates of HEK 293-F cells expressing cytosolic CAT(E) or mitochondrial CAT (F) and those expressing 3MST were mixed and incubated with 2 mM L-cysteine, 0.5 mM �-KG in the presence of Ca2�. *, p � 0.05; **,p � 0.01 versus Ca2� free control. All results are indicated as means � S.E. of at least three experiments.

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H2Swith 3MP as a substratewas not changed byCa2� (Fig. 2D).This observation suggests that the activity of 3MST is not reg-ulated by Ca2�. Because 3MP is unstable and difficult to mea-sure, the amount of H2S produced from cysteine and �-KG bycombined 3MST and CAT was measured to evaluate the Ca2�

dependence of CAT. The production of H2S was decreased byCa2� in a concentration-dependent manner (Figs. 2E and 2F).These observations suggest that the activity of CAT is regulatedby Ca2�.H2S Suppresses Ca2� Influx by Activating V-ATPase—High

concentrations of K� evokes Ca2� influx in photoreceptor cells(29). Because H2S regulates Ca2� channels in astrocytes (11),the effect of H2S on Ca2� influx evoked by high K� was exam-ined. Na2S, a donor of H2S, suppressed Ca2� influx in the outernuclear layer (ONL) (Fig. 3, A–C). Na2S alone did not induceCa2� influx. A similar observation was made in the outer plex-iform layer (OPL) (Fig. 3D). In contrast, the photoreceptorouter segment only weakly responded to high-K�, and theresponses were not affected by Na2S (Fig. 3E). These observa-tions suggest that H2S may regulate voltage-dependent Ca2�

influx in cells of ONL and OPL.The center-surround organization of receptive field is one of

the most important characteristics of the retinal neurons. Thenegative feedback from horizontal cells to photoreceptor cellsplays an important role in the center-surround organization(30). Feedback from horizontal cells to photoreceptor cells ismediated by the suppression of L-type Ca2� channels on pho-toreceptor cells by protons released from V-ATPase on hori-zontal cells (31–34). V-ATPase has two cysteine residues at theATP-binding site of catalytic subunit and reversible formationof disulfide bond results in inactivation of the V-ATPase (35).Because H2S has reducing ability, it can activate the V-ATPaseby reducing the disulfide bond in the catalytic site. We there-

fore examined the type of calcium channels mediating Ca2�

influx and the involvement of V-ATPase in H2S-induced sup-pression of Ca2� influx. Inhibitors specific to L-type voltagegated calcium channels, nifedipine and diltiazem, greatly sup-pressedCa2� influx (Fig. 4A), indicating that L-typeCa2� chan-nels are activated by high K� in agreement with the previousstudy (29, 36, 37). Because protons released from V-ATPasecause acidification, whether or not acidification suppressesCa2� influx was examined. The acidification from pH 7.4 to pH7.2 suppressed the Ca2� influx (Fig. 4B). This observation sug-gests that V-ATPase mediates H2S-induced suppression ofCa2� influx.To confirm the involvement of V-ATPase, the suppressing

effect of H2S on Ca2� influx was investigated in the presence ofinhibitors of V-ATPase, bafilomycin A1 andN-ethylmaleimide(NEM) (34). 100 nM bafilomycin A1, a specific inhibitor of aV-ATPase, abolished the suppressing effect of H2S (Fig. 4C).Five hundred �M NEM, which masks SH-groups at the activesite of V-ATPase (35), also abolished the suppressing effect ofH2S on Ca2� influx (Fig. 4D). These observations suggest thatH2S activates V-ATPase on horizontal cells to release protonsthat suppress L-type Ca2� channels on photoreceptor cells.It was reported that GABA mediates the feedback signal

from horizontal cells to photoreceptor cells (38). We examinedwhether or not GABA mediates the suppressing effect of H2Son Ca2� influx using inhibitors specific for GABA receptors,picrotoxin and bicuculline. Neither picrotoxin nor bicucullinechanged the suppressing effect of H2S on Ca2� influx (Fig. 4, Eand F). These observations suggest that GABAergic input is notinvolved in the suppressing effect of H2S on Ca2� influx.H2S Protects Retinal Photoreceptor Cells from Light-induced

Degeneration—The retina is susceptible to oxidative stressbecause of its high consumption of oxygen and daily exposure

FIGURE 3. H2S suppresses the high K�-evoked Ca2� influx in the outer nuclear layer. A, Ca2� influx was suppressed by H2S. Na2S (10 �M) were added 3 minbefore and during 30 mM K� stimulation. The fluorescence signals were recorded from the outer nuclear layer. B, calcium Green-1 AM fluorescence wasmeasured on �100 �m2 square regions (n 60 – 80) from retinal slices (n 3– 4 slices). ***, p � 0.001. C, suppressing effect of H2S on Ca2� influx. The sensitivityof Ca2� influx to H2S is different depending on regions in the retina. ***, p � 0.001. D, Ca2� influx was suppressed by H2S in the outer plexiform layer. CalciumGreen-1 AM fluorescence was measured on �100 �m2 square regions (n 30 – 40) from retinal slices (n 3– 4 slices). ***, p � 0.001. E, Ca2� influx was notsuppressed by H2S in retinal photoreceptor outer segment. Na2S (10 �M) were added 3 min before and during 30 mM K� stimulation. Error bars indicate S.E.

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to light. Excessive light exposure leads to photoreceptor degen-eration in the retina (39). Photoreceptor cell death is an irre-versible injury that is caused by various factors, such as reactiveoxygen species (ROS) and elevated intracellular concentrationsof Ca2� (40). Because H2S protects neurons from oxidativestress (17, 18, 21), it is possible that H2S protects photoreceptorcells from light-induced retinal degeneration. To examine thispossibility, we investigated the effect of H2S on retinal degen-eration caused by exposing the retina to excessive levels of light.The cytoprotective effect of H2S in vivo was investigated withNaHS, and NaHS as well as Na2S suppressed Ca2� influx in theretina (Fig. 5, A–C) (18). For these reasons we administeredNaHS tomice and examined its effect on photoreceptor degen-eration induced by light exposure. The photoreceptor outersegments were deteriorated (Fig. 5, E and H). In contrast, thelight-induced damage was significantly suppressed in miceadministered NaHS (Fig. 5, F and I). Many cells in the outer

nuclear layer, in which rod inner segments are located, becameTUNEL-positive following exposure to light (Fig. 5, K and N).The administration of NaHS decreased the number of TUNEL-positive cells by�80% relative to a controlwithout any effect onthe density of cells (Fig. 5, L and O–Q).

To confirm the protective effects of H2S on the retinal neu-rons from light-induced degeneration, we examined the levelsof 8-hydroxy-2�-deoxyguanosine (8-OHdG),which is a productof DNA damaged by ROS. We used immunohistochemistrywith an antibody against 8-OHdG. Light exposure produced alot of 8-OHdG positive cells in the outer nuclear layer (Fig. 5T).In contrast, the number of cells positive to 8-OHdG wasdecreased in NaHS-treated mice (Fig. 5V). These observationssuggest that H2S protects photoreceptor cells from light-in-duced retinal degeneration and oxidative stress.

DISCUSSION

The Localization of 3MST and CAT in the Retina—Both3MST and CAT were localized to the retinal neurons, whileCBS and CSE were not found, and H2S was not produced fromcysteine in the absence of �-KG (Figs. 1, B—U and 2A) (41, 42),indicating that the 3MST along with CAT can produce H2S inthe retina. Although both CBS andCSEwere found in salaman-der retina (42), the 3MST/CAT pathway is a major pathway toproduce H2S in mammalian retina.Ca2� Regulation on 3MST/CAT Pathway—The range of

intracellular Ca2� is shifted to the lower concentrations in theretinal neurons compared with the other types of cells in whichthe intracellular concentrations of Ca2� are between 100 nMand 1–2 �M (43). In brightness, the voltage-gated Ca2� chan-nels are closed, and the intracellular concentrations of Ca2� inphotoreceptor cells are reduced to less than 10 nM.When pho-toreceptor cells are hyperpolarized in brightness, a release ofglutamate fromphotoreceptor terminals is decreased, resultingin horizontal cells being in a quiescent state. The intracellularconcentrations of Ca2� in horizontal cells are maintained�50–75 nM in the resting state (44). In darkness, Ca2� ionsenter the photoreceptor cells, and the intracellular concentra-tions ofCa2� reach�600 nM (28). The present study shows thattheH2S-producing activity of 3MST/CATpathway is increasedat low concentrations of Ca2� that is achieved in brightness(Fig. 2B).The present study shows that Ca2� regulates the CAT activ-

ity but that calmodulin is not involved in the regulation (Fig. 2,C,E, and F). The regulation byCa2�without the involvement ofcalmodulin has been demonstrated for three mitochondrialdehydrogenases; pyruvate dehydrogenase, NAD-isocitratedehydrogenase and oxoglutarate dehydrogenase (24). Theseenzymes do not have any Ca2� binding domain, but they arestimulated by Ca2�. The other example is serine racemase,which is a PLP-dependent enzyme similar to CAT, is activatedby Ca2� (45). In this case Ca2� binds to the site formed by twocarboxylate-containing residues (glutamate and aspartate) (45,46). No such Ca2� binding site has been identified in CAT.H2S Regulates Ca2� Influx—The center-surround organiza-

tion is caused by the inhibitory feedback from horizontal cells(30). The proposed mechanisms for the feedback are thechanges in pH in the synaptic cleft and the involvement of

FIGURE 4. H2S suppresses Ca2� influx by activating V-ATPase. A, Ca2�

influx was suppressed by inhibitors of L-type Ca2� channels. Nifedipine (20�M) and diltiazem (100 �M) suppressed Ca2� influx. (n 72 regions from 3retinal slices). **, p � 0.01. B, Ca2� influx was suppressed under acidic condi-tions. High K� stimulation was applied with bicarbonate buffer saline (BBS) atpH7.4 and pH7.2. (n 96 regions from 4 retinal slices). ***, p � 0.001. C, effectof H2S on Ca2� influx was abolished by bafilomycin A1 (Baf A1). Baf A1 (100nM) was added 3 min before and during high K� stimulation. (n 96 –120regions from 4 –5 retinal slices). ***, p � 0.001. D, effect of H2S on Ca2� influxwas abolished by N-ethylmaleimide (NEM). NEM (0.5 mM) was added 6 minbefore high K� stimulation. (n 72 regions from 3 retinal slices). E and F,suppressing effect of H2S on Ca2� influx in the outer nuclear layer was notaffected by picrotoxin or bicuculline. Picrotoxin (100 �M) (E) or bicuculline (20�M) (F) was added 3 min before and during high K� stimulation. n 72regions from retinal slices (n 3 slices). ***, p � 0.001. The fluorescencesignals were recorded from the outer nuclear layer. Error bars indicate S.E.

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GABAergic neurons (31–34, 38). The present study shows thatH2S activates V-ATPase to suppress high K� evoked Ca2�

influx mediated by L-type Ca2� channels (Fig. 4, A–D). Theinfluence of pH changes caused by Na2S and NaHS on the sup-pression of Ca2� influx is negligible for the following reasons. 1)Because bicarbonate buffered saline (BBS) contains 22.6 mM

NaHCO3 continuously bubbled with 95% (v/v) O2 and 5% (v/v)CO2, concentrations less than 10�MNa2S orNaHS do not haveany effect on pH. 2) Na salts shift pH, if any, to alkaline, whilesuppression of Ca2� influx occurs by acidification. Becausephotoreceptor cells and horizontal cells have 3MST and CATto produce H2S, it is possible that H2S activates V-ATPase in

FIGURE 5. H2S protects retinal neurons from light-induced degeneration. A, Ca2� influx was suppressed by H2S. NaHS (10 �M) were added 3 min before andduring 30 mM K� stimulation. The fluorescence signals were recorded from the outer nuclear layer (ONL). Calcium Green-1 AM fluorescence was measured on�100 �m2 square regions (n 60 – 80) from retinal slices (n 3– 4 slices). ***, p � 0.001. B, Ca2� influx was suppressed by H2S in the outer plexiform layer. ***,p � 0.001. C, Ca2� influx was not suppressed by H2S in retinal photoreceptor outer segment. D—I, H&E staining of the retina exposed to great intensity of light.Cross sections of non-treated- (D), light exposure plus vehicle (PBS) (E), light exposure plus NaHS treated- (F) retina at 24 h after light exposure to mice. Squareareas in D, E, and F were magnified and shown in G, H, and I, respectively. J—O, H2S decreased the number of TUNEL-positive cells generated by the lightexposure. Cross sections of non-treated- (J), light exposure plus vehicle (K), light exposure plus NaHS treated- (L) retina at 24 h after light exposure to mice.TUNEL-positive cells (arrowheads) are observed in the ONL as dark brown precipitates. Square areas in J, K, and L were magnified and shown in M, N, and O,respectively. P, quantitative analysis of the total cells in the ONL. Data are represented as means � S.E., n 5 eyes. Q, quantitative analysis of the TUNEL-positivecells in the ONL. Data are represented as means � S.E., n 5 eyes. ***, p � 0.001. R–W, H2S suppresses the levels of 8-OHdG in the retinal neurons induced bylight exposure. The immunohistochemical analysis with an antibody against 8-OHdG in the retina obtained from mice without exposure to light (R); PBS-treated mice 24 h after light exposure (T); and NaHS-treated mice with light exposure (V) are shown. Nuclei were stained by Hoechst 33342 (S, U, W,respectively).

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horizontal cells to release protons to the synaptic cleft that sup-press Ca2� channels in photoreceptor cells.

GABA receptor inhibitors did not affect the suppression ofCa2� influx induced by H2S (Fig. 4, E and F). GABAergic inputfrom horizontal cells to cone photoreceptor cells is present butits contribution is weak and limited, and picrotoxin could notcompletely suppress the cone receptive field surround (32, 47).These observations suggest that GABA may not mediate H2S-induced suppression of Ca2� influx.Ca2� Homeostasis and Photoreceptor Damage—H2S sup-

presses the elevation of Ca2� in the photoreceptor cells by acti-vating V-ATPase in horizontal cells andmaintains Ca2� home-ostasis (Figs. 3 and 4). Intracellular concentrations of Ca2� areincreased during photoreceptor apoptosis, but the L-type Ca2�

channel blocker diltiazem prevents light-induced photorecep-tor cell death (48). The lack of the V-ATPase a3 subunit causesretinal degradation in mice (49). These observations suggestthat the failure ofCa2�homeostasis, which is regulated byCa2�

channels and V-ATPase, by excessive levels of light may causeretinal cell degeneration. The regulation of Ca2� and cytopro-tective effect of endogenous H2S may fail when photoreceptorcells are exposed to excessive levels of light. Even under suchconditions the administration of H2S protects cells from light-induced degeneration.

CONCLUSION

H2S is produced by 3MST alongwith CAT in retinal neuronsand may modulate the negative feedback from horizontal cellsto photoreceptor cells by regulating Ca2� influx through theactivation of V-ATPase. Because H2S protects retinal neuronsfrom light-induced degeneration, the enhancement of 3MST/CAT pathway or the administration of H2S may have clinicalbenefit for diseases with retinal cell degeneration.

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H2S Prevents Light-induced Retinal Degeneration

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Yamada and Hideo KimuraYoshinori Mikami, Norihiro Shibuya, Yuka Kimura, Noriyuki Nagahara, Masahiro

Influx2+Modulation of CaHydrogen Sulfide Protects the Retina from Light-induced Degeneration by the

doi: 10.1074/jbc.M111.298208 originally published online September 20, 20112011, 286:39379-39386.J. Biol. Chem. 

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