犬视网膜毛细血管周细胞和内皮细胞中的多元醇生成及NADPH依赖性还原酶

Polyol formation and NADPH-dependent reductases in dog retinal capillary pericytes and endothelial cells.

作者信息

Sato S, Secchi E F, Lizak M J, Fukase S, Ohta N, Murata M, Tsai J Y, Kador P F

机构信息

Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-1850, USA.

出版信息

Invest Ophthalmol Vis Sci. 1999 Mar;40(3):697-704.

PMID:10067973

Abstract

PURPOSE

Dogs fed a diet containing 30% galactose experience retinal vascular changes similar to those in human diabetic retinopathy, with selective pericyte loss as an initial lesion. In the present study the relationship among reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductases, polyol formation, and flux through the polyol pathway in cultured dog retinal capillary cells were investigated.

METHODS

Pericytes and endothelial cells were cultured from retina of beagle dogs. NADPH-dependent reductases were characterized by chromatofocusing after gel filtration. Sugars in cultured cells were analyzed by gas chromatography, and flux through the polyol pathway was investigated by 19F nuclear magnetic resonance (NMR) with 3-fluoro-3-deoxy-D-glucose (3FG) as a substrate. The presence of aldose reductase and sorbitol dehydrogenase in these cells was examined by northern blot analysis.

RESULTS

Two distinct peaks corresponding to aldose reductase and aldehyde reductase, the latter being dominant, were observed in pericytes by chromatofocusing. Culture in medium containing either 10 mM D-galactose or 30 mM D-glucose resulted in the accumulation of sugar alcohol in pericytes that was markedly reduced by aldose reductase inhibitors. 19F NMR spectra obtained from pericytes cultured for 5 days in medium containing 2 mM 3FG displayed the marked accumulation of 3-fluoro-deoxysorbitol but not 3-fluoro-deoxyfructose. No 3FG metabolism was observed in similarly cultured endothelial cells. With northern blot analysis, aldose reductase was detected in pericytes but not in endothelial cells. Sorbitol dehydrogenase was below the detectable limit in pericytes and endothelial cells.

CONCLUSIONS

Aldose, aldehyde, and glyceraldehyde reductases are present in dog retinal capillary pericytes, with aldehyde reductase being the major reductase present. Polyol accumulation easily occurs in pericytes but not in endothelial cells.

摘要

目的

喂食含30%半乳糖饮食的狗会出现类似于人类糖尿病视网膜病变的视网膜血管变化，最初的病变是选择性周细胞丢失。在本研究中，对培养的狗视网膜毛细血管细胞中还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）依赖性还原酶、多元醇形成以及通过多元醇途径的通量之间的关系进行了研究。

方法

从比格犬的视网膜中培养周细胞和内皮细胞。凝胶过滤后通过色谱聚焦对NADPH依赖性还原酶进行表征。通过气相色谱分析培养细胞中的糖类，并以3-氟-3-脱氧-D-葡萄糖（3FG）为底物，通过19F核磁共振（NMR）研究通过多元醇途径的通量。通过Northern印迹分析检测这些细胞中醛糖还原酶和山梨醇脱氢酶的存在。

结果

通过色谱聚焦在周细胞中观察到两个分别对应醛糖还原酶和醛还原酶的不同峰，后者占主导。在含有10 mM D-半乳糖或30 mM D-葡萄糖的培养基中培养导致周细胞中糖醇积累，醛糖还原酶抑制剂可使其显著减少。在含有2 mM 3FG的培养基中培养5天的周细胞获得的19F NMR光谱显示3-氟脱氧山梨醇明显积累，但未显示3-氟脱氧果糖积累。在同样培养的内皮细胞中未观察到3FG代谢。通过Northern印迹分析，在周细胞中检测到醛糖还原酶，但在内皮细胞中未检测到。山梨醇脱氢酶在周细胞和内皮细胞中低于可检测限。