Mitochondrial oxidative stress has been reported as the result of respiratory complex anomalies, genetic defects, or insufficient oxygen or glucose supply. Although Ca(2+) has no direct effect on respiratory chain function or oxidation/reduction process, mitochondrial Ca(2+) overload can lead to reactive oxygen species (ROS) increase. Even though Ca(2+) is well known for its role as crucial second messenger in modulating many cellular physiological functions, Ca(2+) overload is detrimental to mitochondrial function and may present as an important cause of mitochondrial ROS generation. Possible mechanisms include Ca(2+) stimulated increase of metabolic rate, Ca(2+) stimulated nitric oxide production, Ca(2+) induced cytochrome c dissociation, Ca(2+) induced cardiolipin peroxidation, Ca(2+) induced mitochondrial permeability transition pore opening with release of cytochrome c and GSH-antioxidative enzymes, and Ca(2+)-calmodulin dependent protein kinases activation. Different mechanisms may exist under different mitochondrial preparations (isolated mitochondria vs. mitochondria in intact cells), tissue sources, animal species, or inhibitors used. Furthermore, mitochondrial ROS rise can modulate Ca(2+) dynamics and augment Ca(2+) surge. The reciprocal interactions between Ca(2+) induced ROS increase and ROS modulated Ca(2+) upsurge may cause a feedforward, self-amplified loop createing cellular damage far beyond direct Ca(2+) induced damage.

Enlarged or giant mitochondria have often been documented in aged tissues although their role and underlying mechanism remain unclear. We report here how highly elongated giant mitochondria are formed in and related to the senescent arrest. The mitochondrial morphology was progressively changed to a highly elongated form during deferoxamine (DFO)-induced senescent arrest of Chang cells, accompanied by increase of intracellular ROS level and decrease of mtDNA content. Interestingly, under exposure to subcytotoxic doses of H 2 O 2 (200 mM), about 65% of Chang cells harbored elongated mitochondria with senescent phenotypes whereas ethidium bromide (EtBr) (50 ng/ml) only reformed the cristae structure. Elongated giant mitochondria were also observed in TGF b1-or H 2 O 2 -induced senescent Mv1Lu cells and in old human diploid fibroblasts (HDFs). In all senescent progresses employed in this study Fis1 protein, a mitochondrial fission modulator, was commonly downexpressed. Overexpression of YFP-Fis1 reversed both mitochondrial elongation and appearance of senescent phenotypes induced by DFO, implying its critical involvement in the arrest. Finally, we found that direct induction of mitochondrial elongation by blocking mitochondrial fission process with Fis1-DTM or Drp1-K38A was sufficient to develop senescent phenotypes with increased ROS production. These data suggest that mitochondrial elongation may play an important role as a mediator in stress-induced premature senescence.

Matrix metalloproteinase (MMP)-9 expression induced by interleukin-1b (IL-1b) was investigated in rat brain astrocyte-1 (RBA-1). Here we report that the mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-jB) pathways participate in the induction of MMP-9 expression by IL-1b. Zymographic, western blotting, and RT-PCR analyses showed that IL-1b increased expression of MMP-9 mRNA and protein, which were inhibited by inhibitors of MEK1/2 (U0126), p38 (SB202190), and JNK (SP600125). In accordance with these findings, IL-1b stimulated phosphorylation of p42/p44 MAPK, p38, and c-Jun N-terminal kinase (JNK), which was attenuated by U0126, SB202190, or SP600125, respectively. Furthermore, this up-regulation of MMP-9 mRNA and protein was blocked by a specific NF-jB inhibitor helenalin.Consistently, IL-1b-stimulated translocation of NF-jB into the nucleus and degradation of inhibitory kappa B-a (IjB-a) was revealed by western blotting and immunofluorescence staining, which was blocked by helenalin, but not by U0126, SB202190, or SP600125. Taken together, these results suggest that in RBA-1 cells, activation of p42/p44 MAPK, p38, JNK and NF-jB pathways is essential for IL-1b-induced MMP-9 gene expression via transcription and translation processes. An increased understanding of the signal transduction pathways involved in IL-1b-induced MMP-9 expression on RBA-1 may be of potential therapeutic value in the treatment of inflammatory disease.

concentrations (30-500 mM) inhibited this activity. Acute exposure of control cf-EPC to 100 mM MGO increased basal cytoplasmic Ca 2þ and this was followed by an increased production of mitochondrial superoxide. These new data suggest that MGO whose production is increased shortly after the onset of hyperglycemia is inducing cf-EPC demise by mechanisms that involve perturbations in intracellular calcium homeostasis and increased production of mitochondrial superoxide. Overexpression of glyoxalase 1 minimizes the effects of MGO.

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