15-hydroperoxy-5-8-11-13-eicosatetraenoic-acid and stearic-acid

We have studied interleukin-1 (IL-1)-stimulated signals and gene expression in mesangial cells (MCs) to identify molecular mechanisms of MC activation, a process characteristic of glomerular inflammation. The JNK1 pathway has been implicated in cell fate decisions, and IL-1 stimulates the Jun N-terminal/stress-activated protein kinases (JNK1/SAPK). However, early postreceptor mechanisms by which IL-1 activates these enzymes remain unclear. Free arachidonic acid (AA) activates several protein kinases, and because IL-1 rapidly stimulates phospholipase A2 (PLA2) activity release AA, IL-1-induced activation of JNK1/SAPK may be mediated by AA release.. MCs were grown from collagenase-treated glomeruli, and JNK/SAPK activity in MC lysates was determined using an immunocomplex kinase assay.. Treatment of MCs with IL-1 alpha induced a time-dependent increase in JNK1/SAPK kinase activity, assessed by phosphorylation of the activating transcription factor-2 (ATF-2). Using similar incubation conditions, IL-1 also increased [3H]AA release from MCs. Pretreatment of MCs with aristolochic acid, a PLA2 inhibitor, concordantly reduced IL-1-regulated [3H]AA release and JNK1/SAPK activity, suggesting that cytosolic AA in part mediates IL-1-induced JNK1/SAPK activation. Addition of AA stimulated JNK1/SAPK activity in a time- and concentration-dependent manner. This effect was AA specific, as only AA and its precursor linoleic acid stimulated JNK1/SAPK activity. Other fatty acids failed to activate JNK1/SAPK. Pretreatment of MCs with specific inhibitors of AA oxidation by cyclooxygenase, lipoxygenase, and cytochrome P-450 epoxygenase had no effect on either IL-1- or AA-induced JNK1/SAPK activation. Furthermore, stimulation of MCs with the exogenous cyclooxygenase-, lipoxygenase-, phosphodiesterase-, and epoxygenase-derived arachidonate metabolites, in contrast to AA itself, did not activate JNK1/SAPK.. We conclude that IL-1-stimulated AA release, in part, mediates stimulation of JNK1/SAPK activity and that AA activates JNK1/SAPK by a mechanism that does not require enzymatic oxygenation. JNK1 signaling pathway components may provide molecular switches that mediate structural rearrangements and biochemical processes characteristic of MC activation and could provide a novel target(s) for therapeutic intervention.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Aristolochic Acids; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Dinoprostone; Enzyme Activation; Enzyme Inhibitors; Fatty Acids, Unsaturated; Glomerular Mesangium; Interleukin-1; JNK Mitogen-Activated Protein Kinases; Leukotrienes; Lipid Peroxides; Mitogen-Activated Protein Kinases; Nephritis; Phenanthrenes; Phosphodiesterase Inhibitors; Phospholipases A; Phospholipases A2; Rats; Signal Transduction; Stearic Acids; Tritium; Vasoconstrictor Agents

A complex mixture of different lipid compounds, including phosphatidylcholine, phosphatidylserine, all trans-retinol, 15(S)-hydroperoxyeicosatetraenoic acid, D-alpha-tocopherol, saturated and unsaturated fatty acids can be separated by reversed phase HPLC by using a C-18, 120 mm x 4 mm, 3 microns particle size column and a step gradient from acetonitrile/water (1:1; v:v) to 100% acetonitrile at a flow rate of 0.8 ml/min. By applying this elution condition, separation of various groups of lipid hydroperoxides and lipid derivatives, each one originating from a different in vitro peroxidized polyunsaturated fatty acid, can be obtained. Simultaneous detection is carried out by a diode array detector at a wavelength accumulation range set up between 195 and 400 nm. The possibility of simultaneously having such a large number of measurements renders this chromatographic method particularly suitable in studies concerning lipid peroxidation where, in addition to the detection of free radical-induced lipid hydroperoxides, data on some key antioxidant molecules, i.e. vitamin A and E, as well as that of structural compounds of biological membranes, i.e. phosphatidylcholine and phosphatidylserine, can be achieved.

Topics: Acetonitriles; Arachidonic Acid; Chromatography, High Pressure Liquid; Fatty Acids, Unsaturated; gamma-Linolenic Acid; Leukotrienes; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Lipids; Membrane Lipids; Oleic Acid; Palmitic Acid; Phosphatidylcholines; Phosphatidylserines; Stearic Acids; Vitamin A; Vitamin E