Lysophospholipids and lysophospholipase D in rabbit aqueous humor following corneal injury

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.