Sammendrag
This study investigates the effects of aliphatic (n-heptane, n-nonane), naphtenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)) and aromatic (methylbenzene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on respiratory burst in human granulocytes, measured as 2,7-dichlorofluorescein diacetate-amplified fluorescence and by electron paramagnetic resonance (EPR) spectroscopy. The chemotactic peptide N-formyl-met-leu-phe (fMLP) and phorbol 12-myristate 13-acetate (PMA), a diacylglycerol analogue, were included for purposes of positive control. Fluorescence was elevated in a concentration dependent manner by C9 hydrocarbons. The C7 hydrocarbons did not stimulate respiratory burst in the concentration range examined. The naphtenic hydrocarbon TMCH showed strongest potential for elevation of respiratory burst, and was then selected for mechanistic studies of this free radical formation. In the absence of extracellular Ca2+, fluorescence in response to TMCH and fMLP was reduced by 77% and 90%, respectively. Preincubation of the granulocytes with the protein kinase C inhibitor bisindolylmaleimide reduced the fluorescence stimulated with TMCH, fMLP and PMA by 82%, 56% and 90%, respectively. The phospholipase C inhibitor U73122 lowered the TMCH- and fMLP activated fluorescence by 87% and 76%. In addition, the TMCH- and fMLP induced fluorescence, after the preincubation with the phospholipase D modulator n-butanol, was lowered by 83% and 52%, respectively. The importance of protein kinase C, phospholipase C and phospholipase D for elevation of respiratory burst were also demonstrated by the EPR experiments using the spin trap DEPMPO. Preincubation with the NADPH oxidase inhibitor diphenyleneiodonium, and diethyldithiocarbamate, which inhibits superoxide dismutase, led to an almost complete reduction of fluorescence in response to TMCH, fMLP and PMA. Preincubation with diethyldithiocarbamate led to the elevation of superoxide adducts of DEPMPO. In conclusion, our findings indicate that TMCH activated respiratory burst is dependent on the Ca2+ dependent phospholipase C, phospholipase D and protein kinase C prior to activation of the NADPH oxidase.
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