Nitrosative stress by cyclosporin A in the endothelium: studies with the NO-sensitive probe diaminofluorescein-2/diacetate using flow cytometry

Background. We have reported previously that exposure of endothelial cells to cyclosporin A (CsA) may result; in the regulation of specific genes, suc h as the endothelial nitric oxide synthase. In the context of endothelial t oxicity, whether this represents an adaptive response to injury at the phys iological level or contributes further to the generation of nitrosative str ess; remains to be investigated. The precise reactive species potentially p roduced by CsA and their capacity to modify proteins functionally are not w ell known. Methods. We assessed the possibility of detecting intracellular nitric oxid e (NO) by flow cytometry with-the cell-permeable probe diaminofluorescein-2 diacetate (DAF-2/DA). Results. CsA increased the intracellular-2 h accumulation of DAF-2T, the ox idized form of DAF-2/DA, in an L-NAME-sensitive process. When CsA was re-ad ded for 2 h to bovine aortic endothelial cells previously pre-incubated wit h CsA for 16 h, an additional increase in the accumulation of DAF-2T was ac hieved. Conclusions. In this work, we provide data showing that:,intracellular NO c an be detected by flow cytometry with DAF-2/DA and suggesting two potential mechanisms (transcriptional and post-translational) for the intracellular accumulation of NO in vascular endothelia! cells exposed to the immunosuppr essant CsA.