Department of Pharmaceutical Health Chemistry, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan.
Prostaglandins Other Lipid Mediat. 2012 Mar;97(3-4):83-9. doi: 10.1016/j.prostaglandins.2012.01.003. Epub 2012 Jan 18.
We previously found that lysophosphatidic acid (LPA)-like activity eliciting Cl(-) currents in Xenopus oocytes is increased in rabbit aqueous humor (AH) following corneal freeze wounds. The purpose of this study was to examine whether actual levels of LPA in AH from wounded eyes are higher than those from control eyes, and to determine the sources and enzymatic pathways of AH LPA in control and wounded conditions. Lysophospholipase D (lysoPLD) activity was measured by the enzymatic determination of choline following incubation of AH samples with exogenous lysophosphatidylcholines (LPCs). The molecular species compositions of LPA and LPC in fresh and incubated AH were determined by liquid chromatography-tandem mass spectrometry. A high, but similar activity of lysoPLD in the samples from both control and freeze-wounded eyes was detected. Its enzymatic properties resemble those of plasma lysoPLD, identified as autotaxin. Levels of LPCs, predominant substrates of lysoPLD in AH, were several times higher in the AH samples from injured eyes than those from the control eyes. Our results suggest that lysoPLD is constitutively released from corneal tissues and/or ciliary body into the AH, with no injury-induced increase in release following freeze-wounding. They also suggest that wound-induced increases in LPA-like biological activity are due to linoleoyl species-rich molecular composition in AH from wounded eyes. A possible mechanism of the altered molecular composition is an increase in the AH concentrations of LPCs, linoleoyl species of which are preferentially converted to corresponding unsaturated LPA by the constitutively active lysoPLD.
我们之前发现，在兔眼房水中，角膜冷冻伤后能引起 Xenopus 卵母细胞中氯离子电流的溶血磷脂酸（LPA）样活性增加。本研究的目的是检测受伤眼房水中的 LPA 实际水平是否高于正常眼房水，并确定正常和受伤条件下房水中 LPA 的来源和酶促途径。通过在含有外源性溶血磷脂酰胆碱（LPC）的 AH 样本中孵育，用酶促法测定胆碱来测量溶脂酶 D（lysoPLD）活性。通过液相色谱-串联质谱法测定新鲜和孵育的 AH 中 LPA 和 LPC 的分子种类组成。在对照眼和冷冻伤眼中，检测到 lysoPLD 的活性较高，但相似。其酶学特性与血浆 lysoPLD 相似，被鉴定为自分泌酶。在受伤眼中的 AH 样本中，LPCs 的水平（lysoPLD 在 AH 中的主要底物）比正常眼中的高几倍。我们的结果表明，lysoPLD 是从角膜组织和/或睫状体持续释放到 AH 中的，冷冻伤后释放没有增加。它们还表明，受伤后 LPA 样生物活性的增加是由于受伤眼中 AH 中富含亚油酸的分子组成。改变的分子组成的可能机制是 AH 中 LPC 浓度的增加，其中亚油酸的浓度被组成性激活的 lysoPLD 优先转化为相应的不饱和 LPA。
