No abstract

The protective role of melanin, either synthetic or derived from a metastatic lung melanoma nodule, was studied in terms of its ability to interact with active oxygen species (O2., H2O2, RO., ROO., etc.). Both melanins showed the ability to react with O2.. The superoxide dismutase-like activity corresponds to 21 and 10 U/mg for synthetic and tumor melanin, respectively. The latter value accounts for about 8% of the superoxide dismutase activity of cultured melanoma cells. Neither type of melanin showed catalase-like or glutathione peroxidase-like activity. Both types of melanin reacted with RO. and ROO. radicals as determined by inhibition of the lipid peroxidation reaction of rat liver homogenates. The spontaneous lipid peroxidation of rat liver homogenate was inhibited up to 90% and 80% by synthetic and tumor melanin with half-maximal effects at 2.5 and 5.5 micrograms melanin/ml, respectively. The 2,2-azo-bis-(2 amidino propane) (AAPH)-initiated lipid peroxidation of rat liver homogenate was inhibited up to 30% and 20% by synthetic and tumor melanin, with half maximal effect at 120 and 500 micrograms melanin/ml, respectively. Both types of melanin were able to protect the in vitro inactivation of glucose oxidase, which occurs in the presence of AAPH-generated radicals.

Oxidative stress has recently been suggested to be a mediator of apoptotic cell death [Buttke and Sandstrom (1994) Immunology Today 15, 7-10], although evidence that this phenomenon is a widespread component of apoptosis is lacking. When rat thymocytes were exposed to the glucocorticoid methylprednisolone (MPS), a progressive increase in intracellular peroxides and a decrease in glutathione (GSH) were observed to accompany the onset of apoptosis. Using Percoll density gradients to isolate subpopulations of thymocytes at different stages of apoptosis, the increase in peroxide content was found to be restricted to apoptotic cells, while a significant depletion of GSH and reduced protein thiol was detected in both pre-apoptotic and fully apoptotic cells. To investigate the biological significance of these redox changes, the free radical spin traps 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-1-oxide (TMPO), and the related nitroxide-radical antioxidant 2,2,6,6-tetramethyl-1-piperidinyl-1-oxyl (TEMPO) were tested as inhibitors of thymocyte apoptosis. The cell shrinkage and DNA fragmentation induced by four different initiators of apoptosis were reduced by each compound. TEMPO inhibition of both etoposide- and MPS-induced thymocyte DNA fragmentation was also found to correlate with an increase in intracellular GSH, providing support for the proposal that its antioxidant properties were responsible for the observed protective activity. We conclude that some form of intracellular oxidation (here measured indirectly by changes in intracellular GSH and peroxide levels) is required during thymocyte apoptosis even when this process is initiated by an agent that does not exert a direct oxidant action.

The kinetics of mitochondrial dysfunction after UVB irradiation in human keratinocytes progressed in a time post-irradiation-dependent manner, and they are closely related to cell death. However, there are certain levels of apoptosis, although low, in the absence of mitochondrial alterations. In addition, our data suggest that ROS play a greater role in keratinocyte UVB damage than reactive nitrogen species.

Mitochondria isolated from rat heart, liver, kidney and brain (respiratory control 4.0-6.5) release NO and H2O2 at rates that depend on the mitochondrial metabolic state: releases are higher in state 4, about 1.7-2.0 times for NO and 4-16 times for H2O2, than in state 3. NO release in rat liver mitochondria showed an exponential dependence on membrane potential in the range 55 to 180 mV, as determined by Rh-123 fluorescence. A similar behavior was reported for mitochondrial H2O2 production by [S.S. Korshunov, V.P. Skulachev, A.A. Starkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15_18.]. Transition from state 4 to state 3 of brain cortex mitochondria was associated to a decrease in NO release (50%) and in membrane potential (24-53%), this latter determined by flow cytometry and DiOC6 and JC-1 fluorescence. The fraction of cytosolic NO provided by diffusion from mitochondria was 61% in heart, 47% in liver, 30% in kidney, and 18% in brain. The data supports the speculation that NO and H2O2 report a high mitochondrial energy charge to the cytosol. Regulation of mtNOS activity by membrane potential makes mtNOS a regulable enzyme that in turn regulates mitochondrial O2 uptake and H2O2 production.

The paradigm that nucleocytoplasmic transport of ions occurs without a diffusional barrier has been challenged by the recent demonstration with patch-clamp techniques of the existence of ion channels in the nuclear envelope of murine zygotes and hepatocytes. This report demonstrates the existence of nuclear ion channels (NIC) in murine ventricular cardiac myocytes. NIC conductance (gamma), calculated from current histogram peaks, was 106-532 pS at 22-36 degrees C. In nucleus-attached patches, replacement of cytoplasmic K+ with Na+ reduced NIC activity within 30 s, suggesting that intranuclear-delimited mechanisms mediate this phenomenon. In excised, inside-out patches K+ was as permeable as Na+ through NIC. NIC activity was observed in 0-4 mM Mg2+ and/or ATP2-, with or without 0-1 mM Ca2+, indicating a minor direct role of these ions. However, in non-responsive excised inside-out patches, NIC activity appeared when the catalytic subunit of the cAMP-dependent protein kinase was applied to the nucleoplasmic side of the patch, in the presence of Mg2+ and ATP2-, indicating an important role for phosphorylation-dependent process(es) in NIC function--an observation supported by the depressing effects of protein kinase inhibitor on responsive NIC. The concept that nucleopore complexes are solely responsible for nucleocytoplamic transport leads to the speculation that these structures are the physical substrate for NIC.

No abstract

No abstract