naphthoquinones and diphenyleneiodonium

NAD(P)H oxidase contributes to the pathogenesis of cancer and cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy and heart failure. Plumbagin, a plant-derived naphthoquinone, has been shown to exert anticarcinogenic and anti-atherosclerosis effects in animals. However, the molecular mechanisms underlying these effects remain unknown. It is possible that the beneficial effect of plumbagin is due to the inhibition of NAD(P)H oxidase. Human embryonic kidney 293 (HEK293) and brain tumour LN229 cells express mainly Nox-4, a renal NAD(P)H oxidase. We have examined the effect of plumbagin on Nox-4 activity in HEK293 and LN229 cells using lucigenin-dependent chemiluminescence assay. Plumbagin inhibited the activity of Nox-4 in a time- and dose-dependent manner in HEK293 and LN229 cells. Production of superoxide in HEK293 cells was inhibited by diphenyleneiodonium (DPI), a NAD(P)H oxidase inhibitor. The superoxide production in HEK293 cells was NADPH- and NADH-dependent indicating that the superoxide was generated by a NAD(P)H oxidase in HEK293 cells, but not by the redox-cycling of lucigenin. Furthermore, plumbagin inhibited the superoxide production in Nox-4 transfected COS-7 cells. These results indicated that plumbagin directly interacted with Nox-4 and inhibited its activity.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Enzyme Inhibitors; Humans; Kidney Neoplasms; NAD; NADP; NADPH Oxidase 4; NADPH Oxidases; Naphthoquinones; Onium Compounds; Plumbaginaceae; Transfection

After capacitation, mammalian spermatozoa accomplish the acrosome reaction (AR), a well-controlled exocytosis process crucial to fertilize mature oocytes that involves several protein kinases such as protein kinase A (PKA), C (PKC), and tyrosine kinase (PTK). Reactive oxygen species (ROS) are involved in both bovine sperm capacitation and AR. Lactate dehydrogenase C4 (LDH-C4) was associated with bovine and mouse sperm capacitation. Our aims were to study the participation of LDH-C4 to contribute with the status redox required for AR and the role of ROS in the regulation of PKA, PKC, and PTK involved in the exocytotic event. Sodium oxamate, an inhibitor of LDH-C4, prevented the AR induced by lysophosphatidylcholine (LPC) or NADH. Hydrogen peroxide promoted and superoxide dismutase (scavenger of superoxide), catalase (scavenger of hydrogen peroxide), diphenyleneiodinum, diphenyliodonium, cibacron blue, and lapachol (inhibitors of NADPH oxidase) prevented the AR, suggesting that ROS and a sperm oxidase are involved in the AR induced by these compounds. Inhibitors of PKA, PKC, and PTK also prevented the AR induced by LPC or NADH, suggesting the involvement of these kinases in the process. These results suggest that LDH-C4 may participate in the regulation of the redox status required to achieve the AR in bovine spermatozoa and that ROS are key elements in the regulation of protein kinases associated with the AR process.

Topics: Acrosome; Acrosome Reaction; Analysis of Variance; Animals; Biphenyl Compounds; Catalase; Cattle; Cyclic AMP-Dependent Protein Kinases; Hydrogen Peroxide; Isoenzymes; L-Lactate Dehydrogenase; Lysophosphatidylcholines; Male; Naphthoquinones; Onium Compounds; Protein Kinase C; Protein-Tyrosine Kinases; Reactive Oxygen Species; Sperm Capacitation; Spermatozoa; Superoxide Dismutase; Triazines

Macrophage-like defense cells (hemocytes) of the pond snail Lymnaea stagnalis generate reactive oxygen intermediates (ROIs) upon contact with non-self, following kinetics similar to those of ROI production by mammalian leukocytes during respiratory burst. In this study, several inhibitors of NADPH-oxidase, the key enzyme of the respiratory burst in mammalian phagocytes, were tested for their effect on oxidative activities [as demonstrated by nitroblue tetrazolium (NBT) reduction and luminol-dependent chemiluminescence (LDCL)] of phagocytosing snail hemocytes. In the presence of diphenylene iodonium, zymosan-stimulated hemocytes of L. stagnalis failed to reduce NBT and showed a markedly reduced LDCL response. Also, compounds that prevent assembly of functional NADPH-oxidase complexes in activated mammalian cells were effective; preincubation of hemocytes with 1,4-naphthoquinone inhibited the LDCL response and NBT reduction upon phagocytic stimulation. Furthermore, coincubation but not preincubation with five different catechol-like phenols inhibited oxidative activities of zymosan-stimulated hemocytes. These results imply similarities in composition and regulation of the ROI-generating mechanisms of both mammalian and snail defense cells. It is postulated that in L. stagnalis hemocytes, (1) NADPH-oxidase activity is responsible for ROI production, (2) an active NADPH-oxidase enzyme complex has to be assembled from putative cytosolic and membrane-associated components, and (3) continuous replacement of active NADPH-oxidase enzyme complexes is necessary to sustain respiratory burst-like oxidative activities during interactions with non-self.

Topics: Animals; Anti-Infective Agents; Catechols; Hemocytes; Luminescent Measurements; Luminol; Lymnaea; NADH, NADPH Oxidoreductases; NADPH Oxidases; Naphthoquinones; Nitroblue Tetrazolium; Onium Compounds; Reactive Oxygen Species; Respiratory Burst; Zymosan