Figure 2. Elevated levels of LysoPLD activity in AH derived from primary open angle glaucoma human patients. A. Details of the demographics for the Cataract control and POAG patients. B. LysoPLD activity (expressed per unit of AH total protein) in the AH of POAG patients (n = 20) revealed a significant (* p, = 0.005) increase relative to age-matched cataract control specimens based on the Wilcoxon Rank Sum test. C. Box-and-whisker plot of the AH LysoPLD activity of glaucoma and cataract controls with box showing median and interquartile range. doi:10.1371/journal.pone.0042627.g002 the HTM cells and an additional immunopositive band at around,65 kDa (Fig. 3D). In vitro inhibition of AH ATX activity by S32826 For this experiment, the water soluble chemical inhibitor- S32826 [4-(Tetradecanoylamino) benzyl phosphonic acid disodi- um salt] which inhibits ATX in the nanomolar range [29], was used to assess inhibition of LysoPLD activity in AH derived from the rabbits in vitro. Ten microgram aliquots of rabbit AH total protein were incubated with different concentrations (5–25 mM) of S32826 at 37uC for 20 minutes prior to determining LysoPLD activity using Amplex Red reagent [27]. While S32826 is reported to have an IC50 in the nanomolar range (9 nM) for purified ATX, and a low IC50 for the phosphodiesterase activity of ATX, the IC50 for inhibition of LPA release from adipocytes is much higher (200 nM) [29]. Therefore, we used mM range drug concentrations Autotaxin and IOP Figure 3. Expression and secretion of ATX by primary cultures of trabecular meshwork cells derived from human and porcine eyes. A. RT-PCR based amplification of total RNA derived from human TM cells confirmed expression of the a, b and c variants of ATX. PCR products were sequenced to confirm identity. Similar observations were noted in TM cells from three different human donors. -RT represents negative controls run without reverse transcriptase. B. Immunofluores- cence localization of ATX in primary cultures of human TM cells reveals a vesicular/punctate distribution, characteristic of a secretory protein. C and D. Human (C) and porcine (D) TM cell lysates (20 mg protein) and conditioned media from TM cells (10 mg protein) show the presence of various isoforms of ATX by immunoblot analysis. Bar: 10 mm. doi:10.1371/journal.pone.0042627.g003 to assess the effect of S32828 in AH samples to account for possible matrix effects of a heterogeneous biological sample. Incubation of rabbit AH with 10 mM S32826 inhibited LysoPLD enzyme activity by 90% (n = 4; p,0.01; based on Student’s t-test, Fig. 4). Figure 4. In vitro inhibition of rabbit AH LysoPLD activity by S32826. To confirm that S32826 inhibits LysoPLD activity of AH, 10 mg of AH protein derived from rabbits was pre-treated with S32826 in vitro for 20 minutes prior to analysis of LysoPLD activity using Amplex Red reagent and fluorimetric detection as described in methods. Rabbit AH LysoPLD activity was inhibited by more than 90% by 10 mM S32826 based on 4 independent observations. The chemical structure of S32826 is shown in the figure inset [29]. doi:10.1371/journal.pone.0042627.g004 PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42627