Kashiwagi Kenji, Iizuka Yoko, Mochizuki Seiichi, Tsumamoto Yuichi, Mishima Hiromu K, Araie Makoto, Suzuki Yasuyuki, Tsukahara Shigeo
Department of Ophthalmology, University of Yamanashi Faculty of Medicine, 1110 Shimokato, Tamaho, Yamanashi, 409-3898, Japan.
Brain Res Mol Brain Res. 2003 Apr 10;112(1-2):126-34. doi: 10.1016/s0169-328x(03)00058-5.
The aim of this study was to compare the effects of hypoxia on nitric oxide synthase (NOS) expression and the production of NO between isolated retinal ganglion cells (RGCs) and retinal glial cells. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine the presence of neuronal NOS mRNA, inducible NOS mRNA, and endothelial NOS mRNAs in the two cell types. RGCs and retinal glial cells were separately cultured under hypoxic (10% O(2)) or control (20% O(2)) conditions. Changes in NOS-mRNA expression were quantified by real-time PCR, and nitrite in the medium was measured up to 96 h of culture. The effects of non-NOS- and iNOS-selective inhibitors on hypoxia-induced release of nitrite in the culture medium were evaluated. RT-PCR revealed the presence of three types of NOSs in the two types of cultured cells. Hypoxic culture conditions significantly changed the expression of all NOS mRNAs in retinal glial cells but not in RGCs. NO production showed significant changes corresponding to those of NOS mRNAs in retinal glial cells but not in RGCs, and both NOS inhibitors significantly reduced hypoxia-induced nitrite release in retinal glial cells. Retinal glial cells but not RGCs may be the major source of NO under hypoxic conditions.
本研究的目的是比较缺氧对分离的视网膜神经节细胞(RGCs)和视网膜神经胶质细胞中一氧化氮合酶(NOS)表达及一氧化氮(NO)生成的影响。采用逆转录聚合酶链反应(RT-PCR)检测两种细胞类型中神经元型NOS mRNA、诱导型NOS mRNA和内皮型NOS mRNA的存在情况。RGCs和视网膜神经胶质细胞分别在缺氧(10% O₂)或对照(20% O₂)条件下培养。通过实时PCR定量分析NOS-mRNA表达的变化,并在培养96小时内检测培养基中的亚硝酸盐。评估了非NOS和诱导型NOS选择性抑制剂对缺氧诱导的培养基中亚硝酸盐释放的影响。RT-PCR显示在两种培养细胞类型中存在三种类型的NOS。缺氧培养条件显著改变了视网膜神经胶质细胞中所有NOS mRNA的表达,但在RGCs中未改变。NO生成在视网膜神经胶质细胞中显示出与NOS mRNA相应的显著变化,但在RGCs中未改变,并且两种NOS抑制剂均显著降低了缺氧诱导的视网膜神经胶质细胞中亚硝酸盐的释放。在缺氧条件下,视网膜神经胶质细胞而非RGCs可能是NO的主要来源。