Cellular localization of the inhibitory action of abruquinone A against respiratory burst in rat neutrophils

Abstract

1. The possible mechanisms of action of the inhibitory effect of abruquinone A on the respiratory burst in rat neutrophils in vitro was investigated.

2. Abruquinone A caused an irreversible and a concentration-dependent inhibition of formylmethionyl-leucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O₂^.−) generation with IC₅₀ values of 0.33±0.05 μg ml⁻¹ and 0.49±0.04 μg ml⁻¹, respectively.

3. Abruquinone A also inhibited O₂ consumption in neutrophils in response to fMLP/CB and PMA. However, abruquinone A did not scavenge the generated O₂^.− in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation.

4. Abruquinone A inhibited both the transient elevation of [Ca²⁺]_i in the absence of [Ca²⁺]_o (IC₅₀ 7.8±0.2 μg ml⁻¹) and the generation of inositol trisphosphate (IP₃) (IC₅₀ 10.6±2.0 μg ml⁻¹) in response to fMLP.

5. Abruquinone A did not affect the enzyme activities of neutrophil cytosolic protein kinase C (PKC) and porcine heart protein kinase A (PKA).

6. Abruquinone A had no effect on intracellular guanosine 3′ : 5′-cyclic monophosphate (cyclic GMP) levels but decreased the adenosine 3′ : 5′-cyclic monophosphate (cyclic AMP) levels.

7. The cellular formation of phosphatidic acid (PA) and phosphatidylethanol (PEt) induced by fMLP/CB was inhibited by abruquinone A with IC₅₀ values of 2.2±0.6 μg ml⁻¹ and 2.5±0.3 μg ml⁻¹, respectively. Abruquinone A did not inhibit the fMLP/CB-induced protein tyrosine phosphorylation but induced additional phosphotyrosine accumulation on proteins of 73–78 kDa in activated neutrophils.

8. Abruquinone A inhibited both the O₂^.− generation in PMA-activated neutrophil particulate NADPH oxidase (IC₅₀ 0.6±0.1 μg ml⁻¹) and the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free system (IC₅₀ 1.5±0.2 μg ml⁻¹).

9. Collectively, these results indicate that the inhibition of respiratory burst in rat neutrophils by abruquinone A is mediated partly by the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways, and by suppressing the function of NADPH oxidase through the interruption of electron transport.

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