섬유주세포에서 Rho Kinase 저해제가 일산화질소의 생성에 미치는 영향
Effect of Rho Kinase Inhibitor on the Production of Nitric Oxide in Trabecular Meshwork Cells
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목적: 섬유주세포에서 Rho kinase (ROCK) 저해제가 일산화질소의 생성과 일산화질소합성효소(endothelial nitric oxide synthase, eNOS)의 발현에 미치는 영향을 알아보고자 하였다. 대상과 방법: 사람의 섬유주세포를 일차배양한 후 ROCK 저해제인 Y-27632를 0 μM, 10 μM, 100 μM의 농도로 노출시킨 후 1일, 3일째에 Griess assay를 이용하여 일산화질소의 생성량을 측정하였다. 노출 1일째에 섬유주단층세포층의 투과도와 교원질 겔의 수축에 미치는 영향을 조사하였고, reverse transcription-polymerase chain reaction (RT-PCR)을 이용하여 eNOS mRNA의 발현 정도를 측정하였다. 결과: 10 μM, 100 μM 농도의 Y-27632는 24시간 노출 후 섬유주단층세포층의 투과도를 농도에 비례하여 유의하게 증가시켰으며(all p=0.001), 교원질 겔을 이완시켰다. 또한 Y-27632는 1일과 3일째 모두 일산화질소의 생성량을 증가시켰으며(각각 p=0.020, 0.001), eNOS mRNA의 발현도 함께 증가하였다(p=0.039). 결론: 섬유주세포에서 ROCK 저해제가 섬유주를 통한 방수유출을 증가시키는 기전에 있어서 일산화질소의 생성 증가가 관여할 것으로 생각된다.
Abstract
Purpose: To investigate the effects of Rho kinase (ROCK) inhibitor on the production of nitric oxide (NO) and expression of endothelial nitric oxide synthase (eNOS) in cultured human trabecular meshwork cells (HTMC). Methods: Primarily cultured HTMC were exposed to 0 μM, 10 μM or 100 μM Y-27632 for 3 days and NO production was assessed using Griess assay. After 24 hours, the effect of Y-27632 on the contraction of collagen matrix and the permeability of the HTMC monolayer was determined. The expression of eNOS mRNA was assessed using reverse transcription-polymerase chain reaction (RT-PCR) and cellular survival with the 3-(4, 5 –dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Results: In HTMC, 10 μM and 100 μM Y-27632 significantly increased NO production after 1 day and 3 days (p = 0.020 and 0.001, respectively). At 1 day after exposure, Y-276320 significantly relaxed the collagen matrix and increased the permeability of the HTMC monolayer (all p = 0.001) and the eNOS mRNA expression (p = 0.039). Conclusions: Increased NO production may play a role in the mechanism of increased trabecular outflow associated with ROCK inhibitor.
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