Specialized protective role of mucosal glutathione in pigmented rabbit conjunctiva.
Specialized protective role of mucosal glutathione in pigmented rabbit conjunctiva.
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粘膜谷胱甘肽对兔色素结膜的特殊保护作用。
DOI：
10.1167/iovs.03-0437
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发表时间：
2003
影响因子：
4.4
通讯作者：
Lee,VincentHL
中科院分区：
文献类型：
--
作者：
Gukasyan,HovhannesJ;Kim,Kwang-Jin;Kannan,Ram;Farley,RobertA;Lee,VincentHL
目的。目的探讨过氧化氢降低兔结膜色素组织活性离子转运速率(Isc)的机制及粘膜谷胱甘肽(GSH)的保护作用。在改良的Ussing小室装置中，使用新鲜摘除的有色兔的结膜组织，评估了哇巴因的Isc和特异性结合特性的变化。测定给药后粘膜和浆膜滴注H2 O2的有效抑制浓度(IC50)。测定外源性H_2O_2在粘膜和浆膜中的消耗率。分别用3 mM粘膜8-BR cAMP、0.5 mM布美尼特浆膜和无Na+重碳酸盐林格液(BRS)浸泡结膜，评价结膜离子转运机制在IC50时粘膜H2 O2引起的Isc变化中的作用。在粘膜或浆膜H_2O_2存在的情况下，观察~H哇巴因与结膜浆膜侧的特异性结合，以评价功能性Na+/K~+-ATPase泵在H_2O_2损伤中的作用。还测定了粘膜内注射GSH和其他还原和非还原物质对氧化剂引起的结膜厚度下降的可能恢复的影响。粘膜和浆膜H_2O_2逐渐降低结膜Isc，呈剂量依赖关系。H2 O2对粘膜的IC50为1.49±0.2 0 mm，而浆膜IC50为10.6±2.0μM。在IC50用粘膜H2 O2处理的结膜组织保留了其最大8-BrcAMP依赖增加的大约50%的Isc。布美他尼特不能使结膜内皮细胞的Isc降低70%以上。当结膜在粘膜和浆膜两边用无钠BRS洗浴时，联合作用是相加的，可使Isc降低95%~99%。在IC50下预先暴露于粘膜H_2O_2的结膜组织，在大约100分钟后恢复到初始H_2O_2前的60%~80%。H-哇巴因与组织浆膜侧的特异性结合，在粘膜或浆膜处理时，其IC50值分别为85%和85%。用5 mM GSH、2 mM GSH-Mee或0.1 mM ebselen处理60分钟后，在IC50下胃黏膜过氧化氢暴露后哇巴因结合分别减少30%、45%和55%。此外，粘膜后用10 mM GSH或5 mM GSH-MEE处理预先暴露于粘膜H_2O_2的结膜组织，可使黏膜侧仅暴露于1.5 mM H_2O_2的结膜组织的哇巴因结合水平恢复30%。相反，在IC50时，血清中H_2O_2引起的结膜Isc或哇巴因结合水平的下降是不可逆转的。[H2 O2]对结膜Isc的粘膜IC50越高，粘膜洗涤液对外源性H2 O2的消耗越快。此外，结膜上皮细胞主动分泌GSH可能有助于减轻粘膜应用H_2O_2造成的损伤。H_2O_2损伤可能直接影响参与主动离子跨结膜转运的重要膜组分(如Na~+，K~+-ATPase)。谷胱甘肽(或其类似物)对主动结膜离子转运的粘膜保护作用可能在…中有用
purpose. To investigate mechanisms of H 2 O 2-induced reduction in rates of active ion transport (I sc) across the pigmented rabbit conjunctival tissue and the protective role afforded by mucosal glutathione (GSH).methods. Changes in I sc and specific binding properties of ouabain were evaluated in a modified Ussing chamber setup, using conjunctival tissues freshly excised from pigmented rabbits. Effective concentrations of H 2 O 2 at which 50% of I sc was inhibited (IC 50) were determined for the mucosal and serosal instillation of the agent. The rate of exogenous H 2 O 2 consumption in the mucosal and serosal bathing fluids was estimated. Mucosal 8-Br cAMP at 3 mM, serosal bumetanide at 0.5 mM, and both mucosal and serosal bathing of the conjunctiva with Na+-free bicarbonated Ringer’s solution (BRS) were used to estimate contributions of conjunctival ion transport mechanisms in I sc changes elicited by mucosal H 2 O 2 at IC 50. Specific binding of 3 H-ouabain to the serosal side of the conjunctiva was estimated in the presence of mucosal or serosal H 2 O 2 to assess the role of functional Na+/K+-ATPase pumps in H 2 O 2 injury. The effect of mucosally instilled GSH and other reductive and nonreductive agents on possible restoration of oxidant-induced decrease in conjunctival I sc was also determined.results. Mucosal and serosal H 2 O 2 decreased conjunctival I sc gradually in a dose-dependent manner. The mucosal IC 50 of H 2 O 2 was 1.49±0.20 mM, whereas the serosal IC 50 was estimated at 10.6±2.0 μM. The rate of H 2 O 2 consumption from mucosal fluid was six times faster than that from serosal fluid. Conjunctival tissues pretreated with mucosal H 2 O 2 at IC 50 retained approximately 50% of their maximum 8-Br cAMP-dependent increases in I sc. Serosal bumetanide did not further reduce the I sc beyond the initial 70% decrease caused by mucosal H 2 O 2. When conjunctiva was bathed with Na+-free BRS on both the mucosal and serosal sides, before or after addition of mucosal H 2 O 2, the combined effects were additive, decreasing I sc by up to 95% to 99%. Mucosal, but not serosal, GSH or reduced l-glutathione mono-ethyl ester (GSH-MEE) superfusion of conjunctival tissues pre-exposed to mucosal H 2 O 2 at IC 50 recovered to 60% to 80% of the initial pre-H 2 O 2 I sc after approximately 100 minutes. The specific binding of 3 H-ouabain to the serosal side of the tissue was inhibited by 85% in the presence of mucosal or serosal treatment with H 2 O 2 at their respective IC 50 values. Pretreatment for 60 minutes with either 5 mM GSH, 2 mM GSH-MEE, or 0.1 mM ebselen, when instilled into the mucosal fluid, resulted in 30%, 45%, or 55% reductions, respectively, in ouabain binding after exposure to mucosal H 2 O 2 at IC 50. Furthermore, mucosal posttreatment with 10 mM GSH or 5 mM GSH-MEE of conjunctival tissues pre-exposed to mucosal H 2 O 2 resulted in a 30% recovery of the ouabain-binding level above that observed in tissues exposed to 1.5 mM H 2 O 2 alone on the mucosal side. By contrast, the decrease in conjunctival I sc or in the ouabain-binding level elicited by serosal H 2 O 2 at IC 50 was irreversible.conclusions. A higher mucosal IC 50 of [H 2 O 2] on conjunctival I sc corresponds to the faster consumption of exogenous H 2 O 2 from mucosal bathing fluid. In addition, actively secreted GSH by conjunctival epithelial cells may help reduce the injury by mucosally applied H 2 O 2. Injury by H 2 O 2 may directly affect vital membrane components (eg, Na+, K+-ATPase) involved in active ion transport across conjunctiva. Mucosal protection by GSH (or its analogues) of active conjunctival ion transport may be useful in …