Antamanide, a Derivative of Amanita phalloides, Is a Novel Inhibitor of the Mitochondrial Permeability Transition Pore
Figure 4
Effect of AA on PTP opening in wild-type or CyP-D knock-out mouse fibroblasts and on CyP-D isomerase activity.
A, B, PTP opening of digitonized wild-type (A) or CyP-D knock-out (B) fibroblasts was measured with the whole-cell CRC assay. Experiments were started by the addition of digitonized cells (not shown) followed by pulses of Ca2+ (5 µM each) and are plotted as in Figure 1B. The use of AA is shown with a red trace. Alamethicin (Alm, 1 µM) was added at the end of each measurement to fully release Ca2+ from intracellular stores. C, the CRC/CRC0 ratio calculated as in Figure 1C indicates that both AA and CsA display an inhibitory effect on the PTP in wild-type, but not in CyP-D knock-out fibroblasts. Results are mean±SD of at least 4 experiments. We analyzed whether each pharmacological treatment increased mitochondrial Ca2+ uptake when compared to control conditions (Ca2+ uptake in the absence of the drug), and found a significant difference (Student's t test analysis; *: p<0.01) between the CRC of mitochondria treated with either AA or CsA, and the CRC of untreated mitochondria, indicating that each of these treatments inhibits the PTP in wild-type fibroblasts. D, isomerase activity assay (see Materials and Methods): after addition of the substrate peptide N-succinyl-Ala-Ala-cis-trans-Pro-Phe-p-nitroanilide (arrow), chimotrypsin cleaves the trans-isomeric form, causing a rise in absorbance. AA (8 µM) and CsA (8 µM) act as inhibitors of the enzymatic activity of CyP-D.