| Ca2+ is necessary for numerous cellular processes ranging from fertilization to cell death. The Ca2+concentration in the cytoplasm, ([Ca2+c]), is maintained at least 103 times lower than the extracellular environment. In addition, organelles act as storage sites for Ca2+, which can sequester or release it to ensure that the cytoplasmic Ca2+ is kept at its normal level. The purpose of this research was to measure Ca2+ responses in the cytoplasm and mitochondria to environmental stress. Here, two strains of Aspergillus nidulans, a filamentous fungus, were constructed by DNA-mediated transformation with the aequorin cDNA. One strain expresses aequorin in the cytoplasm whereas the other expresses aequorin in the mitochondria. The aequorin precursor, apoaequorin, is reconstituted with its prosthetic group, coelenterazine. A Ca2+ standard curve was constructed under conditions that compare closely to the internal environment of A. nidulans. Both [Ca2+ c] and mitochondrial Ca2+ concentration ([Ca2+ m]) in unstimulated cells were below the level of detection (<50 nM). The response to different stimuli varied with each stimulus. Ca2+m responses to H2O2 are concentration dependent whereas Ca2+c was not. To examine the source of Ca2+ involved in oxidative stress, the strains were exposed to various inhibitors of Ca2+ channel pumps and channel blockers. Ruthenium red, an inhibitor of mitochondrial Ca 2+ uniporter, inhibited the Ca2+ response to H 2O2 whereas the presence of neomycin, an inhibitor of a mitochondrial Ca2+ release channel, the response was increased. The results indicate that [Ca2+c] and [Ca2+ m] are independent of each other. Oxidative stress activates mitochondrial dehydrogenases and affects ATP concentration. Using mitochondrial and cytosolic cell fractions, ATP levels, superoxide dismutase (SOD) and isocitrate dehydrogenase (IDH) activities were measured. ATP levels decreased with increased exposure to H2O2. SOD activity remained the same whereas IDH activity increased. Cell survival experiments indicated that A. nidulans was able to recover from 100 mM H2O2, but growth was inhibited when cells were pretreated with ruthenium red. These results indicate that A. nidulans has protective mechanisms to combat the effects of oxidative stress that may be regulated by [Ca 2+]. The results and their significance are discussed. |
