Cammarata P R, Chen H Q
Department of Anatomy and Cell Biology, University of North Texas Health Science Center at Fort Worth/North Texas Eye Research Institute 76107.
Invest Ophthalmol Vis Sci. 1994 Mar;35(3):1223-35.
The nature of the association between attenuated myo-inositol-concentrating capability, intracellular polyol accumulation, and hypertonicity-enhanced myo-inositol uptake was investigated in cultured bovine lens epithelial cells (BLECs) exposed to high ambient galactose.
The kinetic characteristics of myo-inositol accumulation based on the measurement of in vitro myo-[3H]inositol (3H-MI) uptake was determined with cultured BLECs incubated in either high ambient galactose or galactose-free, physiological medium under experimental conditions that included both aldose reductase inhibition and elevation of extracellular osmotonicity.
The uptake of 3H-MI was lowered after chronic (20 hour) preincubation of cultured BLECs in 40 mmol/l galactose (i.e., conditions that would favor galactitol synthesis) compared with control cells in galactose-free, physiological medium. Acute exposure (3 hours) of cultured BLECs to a range of 10 to 40 mmol/l galactitol or 5.5 to 44 mmol/l galactose plus the aldose reductase inhibitor (ARI), sorbinil, established by Dixon plot that galactitol, but not galactose, inhibited both the high- and the low-affinity MI transport sites. MI uptake was markedly stimulated in cultured BLECs exposed to galactose-free, hyperosmotic medium by the addition of extracellular raffinose, mannitol, or sorbitol for 20 hours. The enhanced uptake involved increase in the maximal velocity without significant change in Km of both the high- and low-affinity MI transport sites, as indicated by Lineweaver-Burk analysis. However, a similar coadministration of 150 mmol/l sorbitol to the 40 mmol/l galactose (Gal) medium significantly increased, but failed to normalize, the MI uptake relative to that observed with galactose-free, physiological medium. The combined administration of 150 mmol/l sorbitol and the ARI, zopolrestat, to Gal significantly exceeded the MI uptake observed with physiological medium. Exposure of BLECs to cycloheximide for 20 hours did not affect MI uptake in cells maintained in 40 mmol/l galactose but inhibited the otherwise enhanced MI uptake in cells maintained in Gal plus 150 mmol/l sorbitol and zopolrestat in the omission versus the inclusion of cycloheximide.
These results suggest that bovine lens epithelial cells respond to hypertonic stress by elevating myo-inositol transport activity. The increase in MI uptake is due to an increase in the number (or, possibly, a change in the transporter turnover rate) of high- and low-affinity, sodium-dependent MI transporters expressed as a result of the osmotic shock stemming from exposure to hypertonic medium.
在暴露于高环境半乳糖的培养牛晶状体上皮细胞（BLECs）中，研究肌醇浓缩能力减弱、细胞内多元醇积累与高渗增强的肌醇摄取之间关联的性质。
基于体外测量肌醇-[³H]肌醇（³H-MI）摄取来确定肌醇积累的动力学特征，将培养的BLECs在高环境半乳糖或无半乳糖的生理培养基中孵育，实验条件包括醛糖还原酶抑制和细胞外渗透压升高。
与在无半乳糖的生理培养基中的对照细胞相比，培养的BLECs在40 mmol/L半乳糖中慢性（20小时）预孵育后（即有利于半乳糖醇合成的条件下），³H-MI的摄取降低。培养的BLECs急性暴露（3小时）于一系列10至40 mmol/L半乳糖醇或5.5至44 mmol/L半乳糖加醛糖还原酶抑制剂（ARI）索比尼尔，通过迪克森图确定半乳糖醇而非半乳糖抑制了高亲和力和低亲和力的MI转运位点。通过添加细胞外棉子糖、甘露醇或山梨醇20小时，在暴露于无半乳糖的高渗培养基中的培养BLECs中，MI摄取明显受到刺激。如Lineweaver-Burk分析所示，增强的摄取涉及高亲和力和低亲和力MI转运位点的最大速度增加，而Km无显著变化。然而，向40 mmol/L半乳糖（Gal）培养基中同时加入150 mmol/L山梨醇，相对于在无半乳糖的生理培养基中观察到的MI摄取，显著增加但未恢复正常。将150 mmol/L山梨醇和ARI佐泊司他联合给予Gal，显著超过了在生理培养基中观察到的MI摄取。将BLECs暴露于环己酰亚胺20小时，对维持在40 mmol/L半乳糖中的细胞的MI摄取没有影响，但在有无环己酰亚胺的情况下，抑制了维持在Gal加150 mmol/L山梨醇和佐泊司他中的细胞中原本增强的MI摄取。
这些结果表明，牛晶状体上皮细胞通过提高肌醇转运活性来应对高渗应激。MI摄取的增加是由于暴露于高渗培养基引起的渗透休克导致高亲和力和低亲和力、钠依赖性MI转运蛋白的数量增加（或者可能是转运体周转速率的变化）。
