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1 대한안과학회지 214 년제 55 권제 3 호 J Korean Ophthalmol Soc 214;55(3): pissn: eissn: = 증례보고 = 섬유주세포에서국소탄산탈수효소억제제가일산화질소의생성에미치는영향에대한연구 홍정흠 김세은 김재우 대구가톨릭대학교의과대학안과학교실 목적 : 섬유주세포에서국소탄산탈수효소억제제가일산화질소의생성에미치는영향과차이를알아보고자하였다. 대상과방법 : 섬유주세포를일차배양한후혈청이포함되지않은배지를이용하여, 1, 1 μm 의도르졸라미드와브린졸라미드에각각 3 일간노출시킨후 Griess assay 를이용하여일산화질소의생성량을측정하였으며, RT-PCR 을이용하여 enos mrna 의발현을조사하였다. 결과 : 1, 1 μm 의도르졸라미드와브린졸라미드는섬유주세포에서일산화질소의생성을유의하게증가시켰으며 (p<.5), enos mrna 의발현을증가시켰다. 도르졸라미드가브린졸라미드에비해더유의하게일산화질소를생성하였으며 enos mrna 의발현도증가시켰다. 결론 : 섬유주세포에서국소탄산탈수효소억제제는일산화질소의생성과발현을증가시켰으며도르졸라미드가브린졸라미드에비해더유의하게일산화질소의생성을증가시켰다. 따라서국소탄산탈수효소억제제는탄산탈수효소저해작용외에또다른작용기전에의해효과를나타낼것으로생각된다. < 대한안과학회지 214;55(3): > 안과영역에서탄산탈수효소의억제는녹내장치료에있어중요한역할을한다. 1 탄산탈수효소억제제의작용기전은섬모체에서방수생성을억제함으로써안압을낮추는것으로알려졌다. 2,3 이러한안압하강작용외에도탄산탈수효소억제제는시신경과망막의안혈류를증가시키는작용도있는것으로알려졌으며 4-6 탄산탈수효소억제제의혈관이완작용은탄산탈수효소의억제와는무관하며, 7 일산화질소 (Nitric oxide, NO) 의생성과관련이있다. 8 Received: Revised: Accepted: Address reprint requests to Jae Woo Kim, MD, PhD Department of Ophthalmology, Daegu Catholic University Medical Center, #33 Duryugongwon-ro 17-gil, Nam-gu, Daegu , Korea Tel: , Fax: jwkim@cu.ac.kr 섬유주세포는녹내장에서방수유출로의조절에중요한역할을하는데, 섬유주의변성으로인해방수유출로의저항이증가되면개방각녹내장을유발할수있는기전이된다. 9,1 자유유리기인 NO는세포의종류에따라세포의생존에다양한역할을나타낼수있으며, 평활근이완효과도나타내는것으로알려졌다. 섬유주세포는형태학적연구와전기생리학적연구에서평활근과유사한성질을가진것으로알려졌으며 11,12 enos (endothelial nitric oxide synthase) 에서생산하는 NO는섬유주를이완시켜방수유출을촉진하는것으로알려졌다 따라서인체의섬유주세포에서도방수생성을억제하는기전이외에탄산탈수효소억제제가 NO의생성을촉진시켜섬유주를이완시킴으로써방수유출을증가시킬가능성이있으나이에대한연구는아직자세히되어있지않다. 본연구에서는사람의섬유주세포를일차배양하여국소탄산탈수효소억제제가섬유주세포에서 NO의생성과 enos 의발현에미치는영향을알아보고임상적으로사용되고있는국소탄산탈수효소억제제들이 NO의생성에미치는영향에대한차이를비교해보고자하였다. 대상과방법 세포배양안구은행에서얻은사후 6시간이내에적출한안구의앞방각에서섬유주를벗겨내어폴리라이신 (Sigma, USA) 으로처리한배양접시에옮긴후항생제 (Gibco, Invitrogen, Carlsbad, CA, USA) 와 15% 우태아혈청 (Hyclone, USA) 이포함된 Dulbecco s modified Eagle s medium 배지 (DMEM, Gibco, Invitrogen, Carlsbad, CA, USA) 를사용하여 5% CO 2 배양기에서초대배양하였다. 섬유주세포가이식된조직편주위로자라나온것을확인한후섬유주조직의이식편을 416

2 - 홍정흠외 : 탄산탈수효소억제제와일산화질소 - 제거하고배양을계속하였으며세포가배양접시에충만해지면 1% 우태아혈청 (Gibco, Invitrogen, Carlsbad, CA, USA) 을포함한배지로 1:3의비율로트립신처리하여계대배양하였다. 약물처리상용의도르졸라미드 (Dorzolamide, Trusopt R, Merck, Whitehouse Station, NJ, USA) 와브린졸라미드 (Brinzolamide, Azopt R, Alcon, Fort Worth, Texas, USA) 를이용하여실험을시행하였다. 24 well 배양접시에 cells/ml의농도로각 well에세포를분주한후 24시간동안배양기에넣어세포를부착시킨후배지를제거하고나서혈청이포함되지않은 DMEM배지를이용하여희석한도르졸라미드와브린졸라미드에각각 3일간노출시켰다. 이때 enos 저해제인.5 mm (N-Nitro-L-arginine methyl ester hydrochloride, Sigma, St. Louis, MO, USA) 에도동시에노출시켰다. MTT assay와 Griess assay 세포의생존에대한효과는세포증식과세포독성의 screening test 로흔히이용되고있는 colorimetric test 의일종인 MTT (3-[4, 5 dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide, Sigma, St Louis, MO, USA) assay를 16,17 이용하였고 NO의생성은 Griess assay를 18 이용하였다. MTT assay는 3일간각농도의도르졸라미드와브린졸라미드에노출시킨세포의배지에 MTT를각 well당 1 μl씩투여한후 4시간동안정치배양한다음염류용액으로씻어낸후 dimethylsulfoxide (Sigma, St. Louis, MO, USA) 를각 well당.5 ml씩넣어 1분이상흔든다음 96-well plate 에 2 μl씩옮겨 spectrophotometer (FLUOstar Optima, BMG Labtech, Offenburg, Germany) 로 57 nm에서흡광도를측정하였다. 이때세포의증식정도는실험군의값을약물처리를하지않은대조군의비로나누어백분율로나타내었다. Griess assay는각농도의도르졸라미드와브린졸라미드에노출시킨세포의배지에동량의 Griess reagent (Sigma, St. Louis, MO, USA) 를섞은후 96-well plate에옮겨 spectrophotometer로 54 nm에서흡광도를측정하였다. 이때표준치를구하기위해 sodium nitrite (Sigma, St Louis, MO, USA) 를단계적으로희석하여사용하였다. enos mrna 발현을측정하기위한 RT-PCR 섬유주세포에서 Trizol (Invitrogen, Carlsbad, CA, USA) 을이용하여 RNA를분리한후분리된 RNA에서 enos mrna 의발현을 RT-PCR을이용해확인하였다. 3일간각 농도의약물에노출시킨후섬유주세포에서분리한 RNA와 Oligo dt primer, Nuclease-free water 를혼합하여만든 RNA denaturation mix를 7 에 5분간 denaturation 시키고얼음에 5분간둔다음 Prime RT premix (Genet bio, Seoul, Korea) 와혼합하여 42 o C에서 1시간, 7 o C 1분간반응시켜 cdna 로합성하였다. 합성한 cdna 에 2XGoTaq Green Master Mix (Promega, Fitchburg, WI, USA) 와 1 pmol의 forward primer (ctg gct ttc cct tcc agt tc, 225 bp), reverse primer (cct tcc aga tta agg cgg ac, 225 bp) 를각각혼합하여 DNAEngine cycler (Bio-Rad, Hercules, CA, USA) 를사용하여 94 에서 5분, 94 3초, 54 o C 3 초, 72 o C 3초로총 3 cycles를시행한후 57 o C에서 5분간반응시켰다. 증폭된 PCR product를 1% agarose gel에전기영동하여 DNA band를 multi Gauge v2.2 (Fujifilm, Tokyo, Japan) 를이용하여분석하였다. 통계적처리 모든실험은 3계대에서 5계대사이의세포를이용하였고대조군은약물처리를하지않은군으로 3회이상시행하였으며, 실험군과대조군의비교는각농도에따라측정한값을평균 ± 표준오차로나타내어 unpaired t-test 를사용하여비교하였으며유의수준은.5% 로정하였다. Survival (%) 결 D 1 D 1 B 1 B 1 ( μm) Figure 1. Effects of topical carbonic anhydrase ihibitors on the survival of trabecular meshwork cells. Both dorzolamide (D) and brinzolamide (B) did not affect cellular survival at concentrations of 1 and 1 μm (p >.5). 과 국소탄산탈수효소억제제가섬유주세포의생존에미치는영향 국소탄산탈수효소억제제를혈청이결핍된상태에서 3일간섬유주세포에노출시켰을때, 도르졸라미드와브린졸라미드모두 1, 1 μm의농도에서는약물처리를하지않은대조군에비하여세포의생존을유의한영향을미치지 417

3 - 대한안과학회지 214 년제 55 권제 3 호 - 1 (A) Dorzolamide 8 1 μm 1 μm Nitrite ( μ M) (B) Brinzolamide 1 μm 1 μm D 1 D 1 D 1+L D 1+L ( μm) Figure 2. Effects of topical carbonic anhydrase ihibitors on the production of nitric oxide in trabecular meshwork cells. Dorzolamide (D) increased NO production in a dose-dependent manner, which was abolished by co-exposed (L) ( p <.5). (C) β-actin Nitrite ( μ M) B 1 B 1 B 1+L B 1+L ( μm) Figure 3. Effects of topical carbonic anhydrase ihibitors on the production of nitric oxide in trabecular meshwork cells. Brinzolamide (B) increased NO production in a dose-dependent manner which was abolished by co-exposed (L) ( p <.5). 않았으나 (p>.5) (Fig. 1), 1 μm의농도에서는세포의생존이두군모두유의하게감소하였다 (p=.1). 따라서본실험은세포의생존에영향을미치지않는 1, 1 μm의농도에서아래의실험을진행하였다. 국소탄산탈수효소억제제가 NO 의생성에미치는영향 국소탄산탈수효소억제제는무혈청상태에서 3일간노출시켰을때도르졸라미드와브린졸라미드모두약물처리를하지않은대조군에비하여농도에비례하여배지에서의 nitrite 생성을각각유의하게증가시켰다 (p<.5) (Figs. 2, 3). 이때 enos 제해제인.5 mm 를동시에노출시켰을때 NO의생성이억제되었으며이는국소탄산탈수효소억제제에의한 NO 생성증가에 enos 가관여함을나타낸다. 이러한국소탄산탈수효소억제제에의한 NO 생성증가는브린졸라미드에비하여도르졸라미드에서더강한것으로나타났다. Figure 4. Effects of topical carbonic anhydrase inhibitors on the activity of enos in trabecular meshwork cells. Exposure to 1, 1 μm dorzolamide (A) or brinzolamide (B) increased enos mrna expression compared to the non-exposed control. β-actin used as internal standard. Relative expression of enos mrna D 1 D 1 ( μm) Figure 5. Effects on the relative expression of enos mrna after exposure to dorzolamide (D) in trabecular meshwork cells. Exposure to 1, 1 μm dorzolamide increased enos mrna expression compared to the non-exposed control B 1 B 1 ( μm) Figure 6. Effects on the relative expression of enos mrna after exposure to brinzolamide (B) in trabecular meshwork cells. Exposure to 1 μm brinzolamide slightly increased enos mrna expression compared to the non-exposed control

4 - 홍정흠외 : 탄산탈수효소억제제와일산화질소 - 국소탄산탈수효소억제제가 enos mrna의발현에미치는영향국소탄산탈수효소억제제를 3일간섬유주세포에노출시켰을때대조군에비하여 enos 의활성이증가되었다 (Fig. 4). 도르졸라미드의경우대조군에비해 1 μm 농도에서부터 enos 의활성이 7% 정도로증가되었으나브린졸라미드의경우 1 μm 농도에서 1% 정도의활성증가가나타나도르졸라미드가브린졸라미드에비해 enos 의활성에미치는영향이더큰것으로나타났다 (Figs. 5, 6). 고찰 본연구의결과는사람의섬유주세포에서국소탄산탈수효소억제제가 enos 의활성을증가시켜 NO의생성을촉진하였으며, 도르졸라미드가브린졸라미드에비해 NO 생성효과가더크다는것을보여주고있다. 섬모체에작용하여방수생성을억제하여안압을하강시키는시신경과망막의혈류를개선함으로써시신경보호효과를나타내는것으로여겨지고있다. 이러한탄산탈수효소억제제는혈관이완작용은 NO가관여할것이라는생각하였으나이산화탄소, ph (acidosis), HCO - 3 와무관하게나타나므로탄산탈수효소억제와는무관한것으로알려졌는데 7 최근의연구에서도르졸라미드에의한섬모체동맥의이완작용에 NO와혈관내피세포가관여하는것으로보고되었다. 19 따라서혈관내피세포와평활근세포의성질을지닌섬유주세포에서도탄산탈수효소억제제가작용하여 NO의생성에관여할가능성이있다. 11,12 본연구는상용의도르졸라미드 ( 분자량 36.9) 와브린졸라미드 ( 분자량 383.5) 를희석하여시행하였는데고농도에서는두약물모두섬유주세포의생존을유의하게감소시켰으므로세포의생존에영향을미치지않는농도인 1, 1 μm의농도로실험을진행하였다. 방수내에서국소탄산탈수효소억제제의농도는 2-2 μg/ml로알려졌으며 2,21 이는본연구에서사용한 1-1 μm의범위에해당한다. 본연구의결과에따르면국소탄산탈수효소억제제는섬유주세포에서 enos 의활성을증가시키며 NO의생성을증가시키는작용이있는것으로나타났다. 따라서혈관내피세포의경우와유사하게탄산탈수효소억제제는섬유주세포에서 NO의생성을촉진할가능성이있으며이러한효과는평활근세포의성질을가진섬유주세포에작용하여섬유주를이완시킴으로써방수유출을증가시킬가능성이있을것임을시사한다. 그러나잘알려진바와같이국소탄산탈수효소억제제의주된작용기전은방수생성의억제이므로임상적효과에대해서는향후여러가지실험조건이나기간에 따른좀더상세한연구와생체내실험도필요할것으로생각된다. 아세타졸라미드와도르졸라미드는제2형탄산탈수효소를억제하며도르졸라미드가좀더선택적으로작용한다. 1,22,23 도르졸라미드는점안수일후에안구후반부로흡수되어시신경혈류를증가시키는데이는혈관주위조직에의해매개되는것으로알려졌다 도르졸라미드의혈관이완작용의기전은아세타졸라미드와는다르다. 즉아세타졸라미드는주로혈관의평활근세포에직접적으로작용하며혈관주위조직이없는경우이완작용을나타내지않는다. 27 따라서탄산탈수효소억제제들은각각다른기전에의해혈관이완작용을나타내는것으로생각되며, 탄산탈수효소의억제와는무관하게혈관이완작용을나타내므로탄산탈수효소억제제의또다른작용기전에대해서는향후보다자세한연구를요한다. 그러나상용화된약제에는보존제와그외다른성분들이함유되어있으므로이에의한영향을배제할수없다는것이본연구의제한점이다. 28,29 따라서순수한탄산탈수효소억제제성분만으로실험을시행하여이를확인할필요가있을것이다. 또한본실험에서사용한두가지농도외에약물의농도에따른 enos 의활성과 NO의생성추이를좀더자세히살펴볼필요가있을것으로생각된다. 결론적으로섬유주세포에서국소탄산탈수효소억제제는 enos 의활성을증가시키고 NO의생성을촉진시켰으며이러한효과는도르졸라미드가브린졸라미드에비해더크게나타났다. 따라서국소탄산탈수효소억제제는방수생성의억제외에섬유주를통한방수유출을증가시킬가능성이있으나향후그기전과임상적효과에대해보다자세한연구가필요할것으로생각된다. REFERENCES 1) Sugrue MF. The preclinical pharmacology of dorzolamide hydrochloride, a topical carbonic anhydrase inhibitor. J Ocul Pharmacol Ther 1996;12: ) Sugrue MF. Pharmacological and ocular hypotensive properties of topical carbonic anhydrase inhibitors. Prog Retin Eye Res 2; 19: ) Martens-Lobenhoffer J, Banditt P. Clinical pharmacokinetics of dorzolamide. Clin Pharmacokinet 22;41: ) Josefsson A, Sigurdsson SB, Bang K, Eysteinsson T. Dorzolamide induces vasodilatation in isolated pre-contracted bovine retinal arteries. Exp Eye Res 24;78: ) Stefa nsson E, Jensen PK, Eysteinsson T, et al. Optic nerve oxygen tension in pigs and the effect of carbonic anhydrase inhibitors. Invest Ophthalmol Vis Sci 1999;4: ) Pedersen DB, koch Jensen P, la Cour M, et al. Carbonic anhydrase inhibition increases retinal oxygen tension and dilates retinal 419

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6 - 홍정흠외 : 탄산탈수효소억제제와일산화질소 - =ABSTRACT= The Effects of Topical Carbonic Anhydrase Inhibitors on Nitric Oxide Production in Trabecular Meshwork Cells Jeung Hum Hong, MD, Se Eun Kim, MD, Jae Woo Kim, MD, PhD Department of Ophthalmology, Catholic University of Daegu School of Medicine, Daegu, Korea Purpose: To investigate and compare the effects of topical carbonic anhydrase inhibitors on the production and expression of nitric oxide in cultured human trabecular meshwork cells (HTMC). Methods: Primarily cultured HTMC were exposed to, 1, and 1 µm dorzolamide and brinzolamide using serum-deprived media for 3 days. Production of nitric oxide was assessed with Griess assay. Expressions of enos mrna were assessed with RT-PCR. Results: Both dorzolamide and brinzolamide increased the production of nitric oxide enos activity (p <.5). Dorzolamide had a more potent effect than brinzolamide on the production of nitric oxide and the expression of enos mrna. Conclusions: Topical carbonic anhydrase inhibitors increased the production of nitric oxide, which was accompanied by increased enos activity. Dorzolamide had a more potent effect than brinzolamide on the production of nitric oxide and expression of enos mrna in HTMC. The increased production of nitric oxide by topical carbonic anhydrase inhibitors involves mechanisms other than carbonic anhydrase inhibition. J Korean Ophthalmol Soc 214;55(3): Key Words: Brinzolamide, Dorzolamide, Nitric oxide, Topical carbonic anhydrase inhibitor, Trabecular meshwork cells Address reprint requests to Jae Woo Kim, MD, PhD Department of Ophthalmology, Daegu Catholic University Medical Center #33 Duryugongwon-ro 17-gil, Nam-gu, Daegu , Korea Tel: , Fax: , jwkim@cu.ac.kr 421

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<31372D30372D313728B1E8C0E7BFEC2DC0CCB1D9BFEC E687770> 대한안과학회지 2013 년제 54 권제 3 호 J Korean Ophthalmol Soc 2013;54(3):496-501 pissn: 0378-6471 eissn: 2092-9374 http://dx.doi.org/10.3341/jkos.2013.54.3.496 = 증례보고 = 섬유주세포에서 Dipyridamole 이활성산소종과산화스트레스에미치는영향 이근우