No abstract

The production of peroxynitrite during 3-morpholinosydnonimine (SIN-1) decomposition can be continuously monitored, with a sensitivity < or = 0.1 microM, from the kinetics of NADH fluorescence quenching in phosphate buffers, as well as in buffers commonly used with cell cultures, like Locke's buffer or Dulbecco's modified Eagle's medium (DMEM-F12). The half-time for peroxynitrite production during SIN-1 decomposition ranged from 14-18 min in DMEM-F12 (plus and minus phenol red) to 21.5 min in Locke's buffer and 26 min in DMEM-F12 supplemented with apotransferrin (0.1 mg/mL). The concentration of peroxynitrite reached a peak that was linearly dependent upon SIN-1 concentration, and that for 100 microM SIN-1 amounted to 1.4 +/- 0.2 microM in Locke's buffer, 3.2-3.6 microM in DMEM-F12 (plus and minus phenol red) and 1.8 microM in DMEM-F12 supplemented with apotransferrin. Thus, the maximum concentration of peroxynitrite ranged from 1.2 to 3.6% of added SIN-1. NADH was found to be less sensitive than dihydrorhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate to oxidation by H2O2, which is produced during SIN-1 decomposition in common buffers. It is shown that peroxynitrite concentration can be controlled (+/-5%) during predetermined times by using sequential SIN-1 pulses, to simulate chronic exposure of cells or subcellular components to peroxynitrite.

The general affinity of the sarcoplasmic reticulum (SR) Ca 2+ -ATPase was examined for three different classes of vanadium coordination complexes including a vanadium(V) compound, pyridine-2,6-dicarboxylatodioxovanadium(V) (PDC-V(V)), and two vanadium(IV) compounds, bis(maltolato)oxovanadium(IV) (BMOV), and an analogue of amavadine, bis(N-hydroxylamidoiminodiacetato)vanadium(IV) (HAIDA-V(IV)). The ability of vanadate to act either as a phosphate analogue or as a transition-state analogue with enzymes' catalysis phosphoryl group transfer suggests that vanadium coordination compounds may reveal mechanistic preferences in these classes of enzymes. Two of these compounds investigated, PDC-V(V) and BMOV, were hydrolytically and oxidatively reactive at neutral pH, and one, HAIDA-V(IV), does not hydrolyze, oxidize, or otherwise decompose to a measurable extent during the enzyme assay. The SR Ca 2+ -ATPase was inhibited by all three of these complexes. The relative order of inhibition was PDC-V(V) > BMOV > vanadate > HAIDA-V(IV), and the IC 50 values were 25, 40, 80, and 325 µM, respectively. Because the observed inhibition is more potent for PDC-V(V) and BMOV than that of oxovanadates, the inhibition cannot be explained by oxovanadate formation during enzyme assays. Furthermore, the hydrolytically and redox stable amavadine analogue HAIDA-V(IV) inhibited the Ca 2+ -ATPase less than oxovanadates. To gauge the importance of the lipid environment, studies of oxidized BMOV in microemulsions were performed and showed that this system remained in the aqueous pool even though PDC-V(V) is able to penetrate lipid interfaces. These findings suggest that the hydrolytic properties of these complexes may be important in the inhibition of the calcium pump. Our results show that two simple coordination complexes with known insulin enhancing effects can invoke a response in calcium homeostasis and the regulation of muscle contraction through the SR Ca 2+ -ATPase.

Besides the well-known inotropic effects of vanadium in cardiac muscle, previous studies have shown that vanadate can stimulate cell growth or induce cell death. In this work, we studied the toxicity to neonatal rat ventricular myocytes (cardiomyocytes) of two vanadate solutions containing different oligovanadates distribution, decavanadate (containing decameric vanadate, V 10) and metavanadate (containing monomeric vanadate and also di-, tetra-, and pentavanadate). Incubation for 24 h with decavanadate or metavanadate induced necrotic cell death of cardiomyocytes, without significant caspase-3 activation. Only 10 microM total vanadium of either decavanadate (1 microM V 10) or metavanadate (10 microM total vanadium) was needed to produce 50% loss of cell viability after 24 h (assessed with MTT and propidium iodide assays). Atomic absorption spectroscopy showed that vanadium accumulation in cardiomyocytes after 24 h was the same when incubation was done with decavanadate or metavanadate. A decrease of 75% of the rate of mitochondrial superoxide anion generation, monitored with dihydroethidium, and a sustained rise of cytosolic calcium (monitored with Fura-2-loaded cardiomyocytes) was observed after 24 h of incubation of cardiomyocytes with decavanadate or metavanadate concentrations close to those inducing 50% loss of cell viability produced. In addition, mitochondrial membrane depolarization within cardiomyocytes, monitored with tetramethylrhodamine ethyl esther or with 3,3',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide, were observed after only 6 h of incubation with decavanadate or metavanadate. The concentration needed for 50% mitochondrial depolarization was 6.5 +/- 1 microM total vanadium for both decavanadate (0.65 microM V 10) and metavanadate. In conclusion, mitochondrial membrane depolarization was an early event in decavanadate- and monovanadate-induced necrotic cell death of cardiomyocytes.