A novel colorimetric assay for the determination of lysophosphatidic acid in plasma using an enzymatic cycling method

Kishimoto, Tatsuya; Matsuoka, Takeshi; Imamura, Shigeyuki; Mizuno, Koji

doi:10.1016/s0009-8981(03)00165-7

Cited by 57 publications

(50 citation statements)

References 35 publications

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“…This method, in which LPA 1 receptor-mediated cell-rounding activity was measured as a quantitative evaluation of the LPA levels in processed extracts from tissue samples, has been confirmed to be a highly sensitive and specific assay for the evaluation of low levels of LPA in extracts (Inoue et al, 2008b;Ma et al, 2009b), because LPA concentration was detectable from 0.15 pmol (equivalent: 1.5 nM) in this assay, showing the higher sensitivity compared with the enzymatic cycling method, another widely used method for LPA determination that is only suitable for concentrations over 100 nM LPA (Kishimoto et al, 2003). It should be noted that the LPA levels in the SCs and DRs of control mice were negligible (0.79 and 1.81 pmol/mg tissue, respectively).…”

Section: Discussionmentioning

confidence: 95%

Evidence for De Novo Synthesis of Lysophosphatidic Acid in the Spinal Cord through Phospholipase A₂ and Autotaxin in Nerve Injury-Induced Neuropathic Pain

Uchida

Nagai

et al. 2010

J Pharmacol Exp Ther

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We previously reported that lysophosphatidic acid (LPA) initiates nerve injury-induced neuropathic pain and its underlying mechanisms. In addition, we recently demonstrated that intrathecal injection of LPA induces de novo LPA production through the action of autotaxin (ATX), which converts lysophosphatidylcholine to LPA. Here, we examined nerve injury-induced de novo LPA production by using a highly sensitive biological titration assay with B103 cells expressing LPA 1 receptors. Nerve injury caused high levels of LPA production in the ipsilateral sides of the spinal dorsal horn and dorsal roots, but not in the dorsal root ganglion, spinal nerve, or sciatic nerve. Nerve injury-induced LPA production reached its maximum at 3 h after injury, followed by a rapid decline by 6 h. The LPA production was significantly attenuated in ATX heterozygous mutant mice, whereas the concentration and activity of ATX in cerebrospinal fluid were not affected by nerve injury. On the other hand, the activities of cytosolic phospholipase A 2 (cPLA 2 ) and calcium-independent phospholipase A 2 (iPLA 2 ) were enhanced, with peaks at 1 h after injury. Both de novo LPA production and neuropathic pain-like behaviors were substantially abolished by intrathecal injection of arachidonyl trifluoromethyl ketone, a mixed inhibitor of cPLA 2 and iPLA 2 , or bromoenol lactone, an iPLA 2 inhibitor, at 1 h after injury. However, administration of these inhibitors at 6 h after injury had no significant effect on neuropathic pain. These findings provide evidence that PLA 2 -and ATX-mediated de novo LPA production in the early phase is involved in nerve injury-induced neuropathic pain.

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Section: Discussionmentioning

confidence: 95%

Evidence for De Novo Synthesis of Lysophosphatidic Acid in the Spinal Cord through Phospholipase A₂ and Autotaxin in Nerve Injury-Induced Neuropathic Pain

Uchida

Nagai

et al. 2010

J Pharmacol Exp Ther

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“…Determination of LPA and S1P Concentration, lysoPLD Activity, and Western Blotting-LPA and S1P concentrations in plasma and serum were determined as described previously (12)(13)(14). lysoPLD activity was determined as described using 14:0 lysophosphatidylcholine as substrate (5).…”

Section: Atx Knock-out Mice-atx Knock-out Mice (Atx ϫ/ϫmentioning

confidence: 99%

Autotaxin Stabilizes Blood Vessels and Is Required for Embryonic Vasculature by Producing Lysophosphatidic Acid

Tanaka

Okudaira

Kishi

et al. 2006

Journal of Biological Chemistry

431

368

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Autotaxin (ATX) is a cancer-associated motogen that has multiple biological activities in vitro through the production of bioactive small lipids, lysophosphatidic acid (LPA). ATX and LPA are abundantly present in circulating blood. However, their roles in circulation remain to be solved. To uncover the physiological role of ATX we analyzed ATX knock-out mice. In ATXnull embryos, early blood vessels appeared to form properly, but they failed to develop into mature vessels. As a result ATX-null mice are lethal around embryonic day 10.5. The phenotype is much more severe than those of LPA receptor knock-out mice reported so far. In cultured allantois explants, neither ATX nor LPA was angiogenic. However, both of them helped to maintain preformed vessels by preventing disassembly of the vessels that was not antagonized by Ki16425, an LPA receptor antagonist. In serum from heterozygous mice both lysophospholipase D activity and LPA level were about half of those from wild-type mice, showing that ATX is responsible for the bulk of LPA production in serum. The present study revealed a previously unassigned role of ATX in stabilizing vessels through novel LPA signaling pathways. Autotaxin (ATX)2 is a motogen-like phosphodiesterase originally isolated from conditioned medium of human melanoma cells (1). Enforced expression of ATX in Ras-transformed NIH3T3 cells greatly enhances their invasive, tumorigenic, and metastatic potentials (2). In addition, enhanced expression of ATX has been demonstrated in various malignant tumor tissues (3). Thus, ATX is implicated in tumorigenic and metastatic potentials of cancer cells. ATX is also expressed in various tissues and is present at high concentration in various biological fluids including plasma, serum, and seminal plasma (4), implying specific roles of ATX in circulation.Recently, ATX was shown to have lysophospholipase D (lysoPLD) activity, which converts lysophosphatidylcholine to a bioactive lysophospholipid, lysophosphatidic acid (LPA) (5, 6). ATX also converts sphingosylphosphorylcholine into another bioactive lysophospholipid, sphingosine 1-phosphate (S1P) in vitro (7). Because LPA and S1P are regulators of cell motility and proliferation in various cell systems, they might be the effectors of the motogenic actions of ATX. LPA and S1P have been shown to have diverse roles in many biological processes that are mediated by G protein-coupled receptors (GPCRs) specific to LPA or S1P; there are five GPCRs for LPA (LPA 1-5 ) and five for S1P (S1P 1-5 ) with a number of putative GPCRs (8). Thus, ATX may exert its functions through these receptors. Indeed, ATX stimulates cell motility of tumor cells through one of the LPA receptors, LPA 1 (9), and ATX positively or negatively modulates cell motility depending on S1P receptor subtypes (7, 10). To uncover the physiological role of ATX and to identify the endogenous product of ATX, we investigated ATX knock-out mice. In this study we show that ATX produces LPA, but not S1P, in circulating blood and that it contributes to blood vess...

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“…The SPC-hydrolyzing activity was determined by incubating recombinant ATX protein (0.2 g/ml) with 2 mM SPC and measuring the liberated choline as described above. To study the effects of the polar headgroup of lysophospholipids on the enzymatic activity of ATX, recombinant mouse or zebrafish ATX (1.0 g/ml), proteins were mixed with 2 mM 18:1 LPC, LPE, LPS, or LPI in the buffer containing 500 mM NaCl, 5 mM MgCl 2 , and 100 mM Tris-HCl (pH 9.0) in the presence of 0.05% Triton X-100 and incubated at 37°C for 3 h. The amount of LPA generated was determined by the colorimetric method as described previously (23). To measure phosphodiesterase activity, recombinant mouse or zebrafish ATX (0.2 g/ml) proteins were incubated at 37°C for 1 h in a buffer containing 500 mM NaCl, 5 mM MgCl 2 , 100 mM Tris-HCl (pH 9.0), 0.05% Triton X-100, and 4 mM p-nitrophenyl thymidine 5-monophosphate (pNP-TMP; Sigma), and production of p-nitrophenol was analyzed by measuring the optical density at 405 nm.…”

Section: Reagentsmentioning

confidence: 99%

Autotaxin Regulates Vascular Development via Multiple Lysophosphatidic Acid (LPA) Receptors in Zebrafish

Yukiura

Hama

Nakanaga

et al. 2011

Journal of Biological Chemistry

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Background: Autotaxin is essential for vascular development in mice, but the underlying mechanism remains unknown. Results: Autotaxin had similar vascular functions in zebrafish. Furthermore, suppression of lysophosphatidic acid receptors (LPA 1 and LPA 4 ) led to similar vascular defects. Conclusion: Autotaxin exerts its vascular functions by activating LPA 1 and/or LPA 4 in zebrafish. Significance: LPA is a critical factor for regulating angiogenesis in vertebrates.

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A novel colorimetric assay for the determination of lysophosphatidic acid in plasma using an enzymatic cycling method

Cited by 57 publications

References 35 publications

Evidence for De Novo Synthesis of Lysophosphatidic Acid in the Spinal Cord through Phospholipase A₂ and Autotaxin in Nerve Injury-Induced Neuropathic Pain

Evidence for De Novo Synthesis of Lysophosphatidic Acid in the Spinal Cord through Phospholipase A₂ and Autotaxin in Nerve Injury-Induced Neuropathic Pain

Autotaxin Stabilizes Blood Vessels and Is Required for Embryonic Vasculature by Producing Lysophosphatidic Acid

Autotaxin Regulates Vascular Development via Multiple Lysophosphatidic Acid (LPA) Receptors in Zebrafish

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A novel colorimetric assay for the determination of lysophosphatidic acid in plasma using an enzymatic cycling method

Cited by 57 publications

References 35 publications

Evidence for De Novo Synthesis of Lysophosphatidic Acid in the Spinal Cord through Phospholipase A2 and Autotaxin in Nerve Injury-Induced Neuropathic Pain

Evidence for De Novo Synthesis of Lysophosphatidic Acid in the Spinal Cord through Phospholipase A2 and Autotaxin in Nerve Injury-Induced Neuropathic Pain

Autotaxin Stabilizes Blood Vessels and Is Required for Embryonic Vasculature by Producing Lysophosphatidic Acid

Autotaxin Regulates Vascular Development via Multiple Lysophosphatidic Acid (LPA) Receptors in Zebrafish

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Evidence for De Novo Synthesis of Lysophosphatidic Acid in the Spinal Cord through Phospholipase A₂ and Autotaxin in Nerve Injury-Induced Neuropathic Pain

Evidence for De Novo Synthesis of Lysophosphatidic Acid in the Spinal Cord through Phospholipase A₂ and Autotaxin in Nerve Injury-Induced Neuropathic Pain