linoleic-acid and 3-nitrotyrosine

It is well known that excessive non-esterified fatty acids in diabetes contribute to the pathogenesis of renal complications although the mechanism remains elusive. Enhanced oxidative stress has been hypothesized as a unified factor contributing to diabetic complications and increased protein nitrotyrosylation has been reported in the kidneys of diabetic patients. In the current manuscript we described that linoleic acid (LA) caused mitochondrial Ca(2+) efflux and peroxynitrite production, along with increased nitrotyrosine levels of cellular proteins in primary human mesangial cells. The peroxynitrite production by LA was found to depend on mitochondrial Ca(2+) efflux. Downregulation of hsp90beta1, which has been previously shown to be essential for polyunsaturated fatty acid-induced mitochondrial Ca(2+) efflux, significantly diminished LA-responsive mitochondrial Ca(2+) efflux and the coupled peroxynitrite generation, implicating a critical role of hsp90beta1 in the LA responses. Our results further demonstrated that mitochondrial complexes I and III were directly involved in the LA-induced peroxynitrite generation. Using the well established type 2 diabetic animal model db/db mice, we observed a dramatically enhanced LA responsive mitochondrial Ca(2+) efflux and protein nitrotyrosylation in the kidney. Our study thus demonstrates a cause-effect relationship between LA and peroxynitrite or protein nitrotyrosylation and provides a novel mechanism for lipid-induced nephropathy in diabetes.

Topics: Animals; Calcium; Diabetes Mellitus, Experimental; Fatty Acids, Unsaturated; Gene Expression Regulation; HSP90 Heat-Shock Proteins; Humans; Immunohistochemistry; Kidney; Linoleic Acid; Mice; Mitochondria; Models, Biological; Peroxynitrous Acid; Tyrosine

Hypertriglyceridemia, an important risk factor of atherosclerosis, is associated with increased circulating free fatty acids. Research to date indicates that linoleic acid (LA), the major fatty acid in the American diet, may be atherogenic by activating vascular endothelial cells. However, the exact signaling mechanisms involved in LA-mediated proinflammatory events in endothelial cells still remain unclear. We previously reported increased superoxide formation after LA exposure in endothelial cells. The objective of the present investigation is to determine the role of calcium and peroxynitrite in mediating the proinflammatory effect of LA in vascular endothelial cells. LA exposure increased intracellular calcium, nitric oxide, and tetrahydrodiopterin levels as well as the expression of E-selectin. Inhibiting calcium signaling using 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and heparin decreased the expression of E-selectin. Also, LA-mediated nuclear factor kappa B activation and E-selectin gene expression were suppressed by Mn (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (a superoxide scavenger), N(G)-monomethyl-l-arginine (an endothelial nitric oxide synthase inhibitor), and 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III) chloride (a peroxynitrite scavenger). LA exposure resulted in increased nitrotyrosine levels, as observed by Western blotting and immunofluorescence. Our data suggest that the proinflammatory effects of LA can be mediated through calcium and peroxynitrite signaling.

Topics: Animals; Borohydrides; Calcium; Calcium Signaling; Cells, Cultured; E-Selectin; Endothelium, Vascular; Gene Expression Regulation; Linoleic Acid; Models, Biological; NF-kappa B; Nitric Oxide; Peroxynitrous Acid; Pulmonary Artery; Swine; Tyrosine

Reactive species formed from nitric oxide (NO) nitrate unsaturated fatty acids such as linoleate (LA) to nitrated derivatives including nitrolinoleate (LNO(2)). The effect of LNO(2) on human platelets was examined to define how nitrated lipids might behave in vivo. LNO(2), but not LA or 3-nitrotyrosine, dose dependently (0.5-10 microm) inhibited thrombin-mediated aggregation of washed human platelets, with concomitant attenuation of P-selectin expression and selective phosphorylation of VASP at the cAMP-dependent protein kinase selective site, serine 157. LNO(2) caused slight mobilization of calcium (Ca(2+)) from intracellular stores but significantly inhibited subsequent thrombin-stimulated Ca(2+) elevations. LNO(2) did not elevate platelet cGMP, and its effects were not blocked with inhibitors of NO signaling (oxyhemoglobin, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. 2-fold elevations in cAMP were found following LNO(2) treatment of platelets, and the adenylyl cyclase inhibitors 2',5'-dideoxyadenosine and SQ22536 partially restored thrombin-stimulated aggregation. Finally, LNO(2) significantly inhibited cAMP hydrolysis to AMP by platelet lysates. These data implicate cAMP in the anti-aggregatory action of LNO(2). The platelet inhibitory actions of LNO(2) indicate that nitration reactions that occur following NO generation in an oxidizing environment can alter the activity of lipids and lend insight into mechanisms by which NO-derived species may modulate the progression of vascular injury.

Topics: 1-Methyl-3-isobutylxanthine; Blood Platelets; Calcium; Calcium Signaling; Cyclic AMP; Cyclic GMP; Humans; In Vitro Techniques; Kinetics; Linoleic Acid; Linoleic Acids; Nitro Compounds; Phosphoproteins; Phosphorylation; Platelet Activation; Platelet Aggregation; Thrombin; Tyrosine; Vasodilator Agents

Peroxynitrite-mediated linoleic acid oxidation and tyrosine nitration were analysed in the presence of synthetic model neuromelanins: dopamine (DA) -melanin, cysteinyldopamine (CysDA) -melanin and various DA/CysDA copolymers. The presence of melanin significantly decreased the amount of 3-nitrotyrosine formed. This inhibitory effect depended on the type and concentration of melanin polymer. It was found that incorporation of CysDA-derived units into melanin attenuated its protective effect on tyrosine nitration induced by peroxynitrite. In the presence of bicarbonate, the melanins also inhibited 3-nitrotyrosine formation in a concentration dependent manner, although the extent of inhibition was lower than in the absence of bicarbonate. The tested melanins inhibited peroxynitrite-induced formation of linoleic acid hydroperoxides, both in the absence and in the presence of bicarbonate. In the presence of bicarbonate, among the oxidation products appeared 4-hydroxynonenal (HNE). CysDA-melanin inhibited the formation of HNE, while DA-melanin did not affect the aldehyde level. The results of the presented study suggest that neuromelanin can act as a natural scavenger of peroxynitrite.

Topics: Aldehydes; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Dose-Response Relationship, Drug; Hydrogen Peroxide; Linoleic Acid; Melanins; Nitrogen; Oxygen; Peroxynitrous Acid; Tyrosine