Gukasyan Hovhannes J, Kannan Ram, Lee Vincent H L, Kim Kwang-Jin
Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, USA.
Invest Ophthalmol Vis Sci. 2003 Mar;44(3):1202-10. doi: 10.1167/iovs.02-0409.
Metabolism and transport of cysteine are critical for maintenance of the intracellular glutathione (GSH) level. In this study, transport mechanisms of L-cystine and regulation of GSH biosynthesis in the absence or presence of NO-induced oxidant stress were investigated in primary cultured rabbit conjunctival epithelial cells (RCECs).
RCECs were grown in membrane filters to exhibit tight barrier properties. Uptake and transepithelial transport of L-cystine were determined in the presence or absence of extracellular Na(+). Uptake was determined at 10 minutes after (14)C-L-cystine instillation into apical (a) or basolateral (b) bathing fluid. The effect of nitric oxide (NO) on L-cystine uptake, cellular GSH level, and expression level of two subunits of the rate-limiting enzyme gamma-glutamylcysteine synthetase (GCS) was examined after a 24-hour incubation of primary cultured RCECs with an NO donor, S-nitroso-N-acetylpenicillamine (SNAP; N-acetyl-3-(nitrosothio)-D-valine.
Cellular uptake of L-cystine by RCECs occurred through both Na(+)-dependent and -independent mechanisms. Uptake from apical fluid was higher than that from basolateral fluid, except for the highest concentration of L-cystine tested (200 microM). Transepithelial permeability (P(app)) of L-cystine (at 2.5 microM) was three times higher in the a-to-b direction than in the b-to-a direction in the presence of Na(+), whereas the reverse was true in the absence of Na(+). Na(+)-dependent L-cystine uptake from apical fluid was significantly elevated in primary cultured RCECs treated for 24 hours with various concentrations (0.1-2.0 mM) of SNAP, with maximum uptake observed at 1 mM. A similar pattern of SNAP-induced increase of Na(+)-independent L-cystine uptake from apical fluid was observed, whereas no significant difference was observed for basolateral uptake. Concomitantly, a significant elevation of intracellular GSH (up to fivefold versus the control) was recorded, with the highest increase occurring at 0.1 to 0.25 mM SNAP. A parallel increase in the expression levels of both catalytic and regulatory subunits of GCS was observed by Western blot analysis of lysates from RCECs treated with 0.25 mM SNAP for 24 hours.
L-Cystine is transported by both Na(+)-dependent and -independent amino acid transport systems in RCECs. At low substrate concentrations, L-cystine uptake was higher from apical than basolateral fluid. Permeability studies indicated net absorption of L-cystine across RCECs. SNAP caused significant increases in both L-cystine uptake and intracellular GSH level, which occurred concomitantly with elevation of both catalytic and regulatory subunits of GCS. Understanding sulfur amino acid precursor-dependent cellular mechanisms of GSH homeostasis would be of value in devising GSH-based treatment for conjunctival or other ocular disorders.
半胱氨酸的代谢和转运对于维持细胞内谷胱甘肽（GSH）水平至关重要。在本研究中，我们在原代培养的兔结膜上皮细胞（RCECs）中研究了L-胱氨酸的转运机制以及在有无一氧化氮（NO）诱导的氧化应激情况下GSH生物合成的调节。
RCECs在膜滤器中生长以表现出紧密的屏障特性。在有或无细胞外Na⁺的情况下测定L-胱氨酸的摄取和跨上皮转运。在将¹⁴C-L-胱氨酸滴入顶端（a）或基底外侧（b）浴液10分钟后测定摄取情况。在用NO供体S-亚硝基-N-乙酰青霉胺（SNAP；N-乙酰-3-（亚硝基硫基）-D-缬氨酸）对原代培养的RCECs进行24小时孵育后，检测NO对L-胱氨酸摄取、细胞内GSH水平以及限速酶γ-谷氨酰半胱氨酸合成酶（GCS）两个亚基表达水平的影响。
RCECs对L-胱氨酸的细胞摄取通过Na⁺依赖性和非依赖性机制发生。除了测试的最高浓度L-胱氨酸（200μM）外，从顶端液的摄取高于从基底外侧液的摄取。在有Na⁺存在的情况下，L-胱氨酸（2.5μM）的跨上皮通透性（P(app)）在a到b方向比在b到a方向高3倍，而在无Na⁺时则相反。用不同浓度（0.1 - 2.0 mM）的SNAP处理原代培养的RCECs 24小时后，顶端液中Na⁺依赖性L-胱氨酸摄取显著增加，在1 mM时观察到最大摄取。观察到SNAP诱导的顶端液中Na⁺非依赖性L-胱氨酸摄取增加的类似模式，而基底外侧摄取未观察到显著差异。同时，细胞内GSH显著升高（与对照相比高达五倍），在0.1至0.25 mM SNAP时升高最高。通过对用0.25 mM SNAP处理24小时的RCECs裂解物进行蛋白质印迹分析，观察到GCS催化亚基和调节亚基的表达水平平行增加。
L-胱氨酸在RCECs中通过Na⁺依赖性和非依赖性氨基酸转运系统进行转运。在低底物浓度下，顶端液中L-胱氨酸的摄取高于基底外侧液。通透性研究表明L-胱氨酸跨RCECs的净吸收。SNAP导致L-胱氨酸摄取和细胞内GSH水平显著增加，这与GCS催化亚基和调节亚基的升高同时发生。了解依赖于硫氨基酸前体的GSH稳态细胞机制对于设计基于GSH的结膜或其他眼部疾病治疗方法具有重要意义。
