Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities that accounts for many biological properties of serum. LPA is thought to be produced during serum formation based on the fact that the LPA level is much higher in serum than in plasma. In this study, to better understand the pathways of LPA synthesis in serum, we evaluated the roles of platelets, plasma, and phospholipases by measuring LPA using a novel enzyme-linked fluorometric assay. First, examination of platelet-depleted rats showed that half of the LPA in serum is produced via a platelet-dependent pathway. However, the amount of LPA released from isolated platelets after they are activated by thrombin or calcium ionophore accounted for only a small part of serum LPA. Most of the platelet-derived LPA was produced in a two-step process: lysophospholipids such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine, and lysophosphatidylserine, were released from activated rat platelets by the actions of two phospholipases,

We previously found hydroperoxide-responsive proteins (HPRPs), which are comprised of peroxiredoxin I (Prx I), Prx II, Prx III, Prx VI, HSP27, G3PDH and two unidentified proteins (HPRP-2' and HPRP-5'), in human umbilical vein endothelial cells. It was demonstrated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) that most HPRPs are converted into variants with lower pI upon exposure to hydroperoxides. In this study, we examined the HPRP response on 2D gels upon exposure of human endothelial cells (ECV304) to paraquat (PQ2+), which generates reactive oxygen species (ROS) within cells. PQ2+ exerted cytotoxic effects in a dose-(10 microM-10 mM) and time-(24-168 h) dependent manner. Two-dimensional PAGE analysis revealed that HPRP-2', and oxidized forms of Prx I, Prx II and Prx III were clearly increased upon exposure of cells to sublethal levels of PQ2+. Microsequence analysis revealed that both HPRP-2 and -2' were identical with human DJ-1. Moreover immunoblot analysis confirmed the increase of oxidized forms of Prx II, Prx III and DJ-1 in response to sublethal levels of PQ2+. PQ2+ treatment failed to increase fluorescence intensity derived from DCF, which is believed to be an indicator for intracellular levels of hydroperoxide. Although pentachlorophenol (PCP), an uncoupler of the mitochondrial respiratory chain, clearly elevated the fluorescence, PCP had no effect on HPRP response. These observations indicated that DCF-derived fluorescence is not correlated with HPRP response. We consider that the response of Prxs and DJ-1 on 2D gels could reflect endogenous production of ROS in PQ(2+)-treated cells, and might be a sensitive indicator of oxidative stress status.

Disrupted-In-Schizophrenia-1 (DISC1) is a unique susceptibility gene for major mental conditions, because of the segregation of its genetic variant with hereditary psychosis in a Scottish pedigree. Genetic association studies reproducibly suggest involvement of DISC1 in both schizophrenia and bipolar disorder in several ethnic groups. The DISC1 protein is multifunctional, and a pool of DISC1 in the dynein motor complex is required for neurite outgrowth in PC12 cells as well as proper neuronal migration and dendritic arborization in the developing cerebral cortex in vivo. Here, we show that a specific interaction between DISC1 and nuclear distribution element-like (NDEL1/NUDEL) is required for neurite outgrowth in differentiating PC12 cells. Among several components of the dynein motor complex, DISC1 and NDEL1 are selectively upregulated during neurite outgrowth upon differentiation in PC12 cells. The NDEL1 binding site of DISC1 was narrowed down to a small portion of exon 13, corresponding to amino acids 802-835 of DISC1. We demonstrate that genetic variants of DISC1, proximal to the NDEL1 binding site, affect the interaction between DISC1 and NDEL1.

Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the halftype ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral

ABCA3 protein is expressed predominantly at the limiting membrane of the lamellar bodies in alveolar type II cells, and mutations in the ABCA3 gene cause lethal respiratory distress in newborn infants. To investigate the function of ABCA3 protein, we generated Abca3-deficient mice by targeting Abca3. Fullterm Abca3؊/؊ newborn pups died within an hour after birth because of acute respiratory failure. Ultrastructural analysis revealed abnormally dense lamellar body-like organelles and no normal lamellar bodies in Abca3 ؊/؊ alveolar type II cells. TLC and electrospray ionization mass spectrometry analyses of lipids in the pulmonary interstitium showed that phosphatidylcholine and phosphatidylglycerol, which contain palmitic acid and are abundant in normal surfactant lipids, were dramatically decreased in Abca3 ؊/؊ lung. These findings indicate that ABCA3 plays an essential role in pulmonary surfactant lipid metabolism and lamellar body biogenesis, probably by transporting these lipids as substrates.

No abstract

In an effort to elucidate the functions of secreted phospholipase A 2 (sPLA 2 ) enzymes in vivo, we generated transgenic (Tg) mice for group V sPLA 2 (sPLA 2 -V) and group X sPLA 2 (sPLA 2 -X), which act potently on phosphatidylcholine in vitro. We found that sPLA 2 -V Tg mice died in the neonatal period because of respiratory failure. The lungs of sPLA 2 -V Tg mice exhibited atelectasis with thickened alveolar walls and narrow air spaces, accompanied by infiltration of macrophages and only modest changes in eicosanoid levels. This severe pulmonary defect in sPLA 2 -V Tg mice was attributable to marked reduction of the lung surfactant phospholipids, phosphatidylcholine and phosphatidylglycerol. Given that the expression of sPLA 2 -V is greatly elevated in human lungs with severe inflammation, our present results raise the intriguing possibility that this isozyme may contribute to ongoing surfactant hydrolysis often observed in the lungs of patients with respiratory distress syndrome. In contrast, sPLA 2 -X Tg neonates displayed minimal abnormality of the respiratory tract with normal alveolar architecture and surfactant composition. This unexpected result was likely because sPLA 2 -X protein existed as an inactive zymogen in most tissues. The active form of sPLA 2 -X was detected in tissues with inflammatory granulation in sPLA 2 -X Tg mice. These results suggest that sPLA 2 -X mostly remains inactive under physiological conditions and that its proteolytic activation occurs during inflammation or other as yet unidentified circumstances in vivo.

Although individual mammalian secreted phospholipase A 2 (sPLA 2 ) enzymes exhibit unique tissue and cellular distributions, the cell type-specific functions of each enzyme remain largely unknown. In this study, we found by immunohistochemistry that group X sPLA 2 (sPLA 2 -X) is uniquely located in the peripheral neuronal fibers, an observation that was supported by detection of its transcript and protein in the neuronal cell line PC12 and in primary dorsal root ganglia neurons. Adenoviral expression of sPLA 2 -X in PC12 cells facilitated neurite outgrowth, particularly when combined with a suboptimal concentration of nerve growth factor. In neuronally differentiated PC12 cells, sPLA 2 -X was preferentially localized in the Golgi apparatus and growth cones, and proteolytic conversion of the proenzyme to mature enzyme mainly occurred after the secretion process. The neurite-extending ability of sPLA 2 -X depended on the production of its catalytic product, lysophosphatidylcholine. Moreover, nerve growth factor-induced neurite extension of PC12 cells was modestly but significantly attenuated by an anti-sPLA 2 -X antibody or by a small interfering RNA for sPLA 2 -X. These observations suggest the potential contribution of sPLA 2 -X to neuronal differentiation, and possibly repair, under certain conditions.