Antamanide, a Derivative of Amanita phalloides, Is a Novel Inhibitor of the Mitochondrial Permeability Transition Pore
Figure 1
Effect of AA on PTP opening in isolated mouse liver mitochondria.
A, chemical structure of AA. B, D, Ca2+ retention capacity (CRC) either in phosphate (Pi) buffer (B) or in arsenate (Asi) buffer (D). Calcium Green-5N fluorescence is reported as arbitrary units on the y axis. As the probe does not permeate mitochondria, Ca2+ uptake into the organelles is displayed as a rapid decrease of the fluorescence spike after administration of every Ca2+ pulse (10 µM each). AA (red trace, 8 µM) or CsA (0.8 µM) act as pore inhibitors only in Pi buffer (B), as they increase the threshold Ca2+ concentration required to trigger the permeability transition, i.e. the number of spikes before a sudden and marked fluorescence increase occurs. Ub0 (25 µM) inhibits the pore also in Asi buffer, albeit to a lesser extent. C, inset of D, quantification of the effect of PTP inhibitors is displayed as the ratio between the CRC detected in the presence (CRC) and absence (CRC0) of the compound. Results are mean±SD of at least 4 experiments. In C and D, 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 (at various concentrations), or CsA, or Ub0 and the CRC of untreated mitochondria, indicating that each of these treatments inhibits the PTP. In C, significant differences were also observed between the CRC of mitochondria treated with either Ub0/AA or Ub0/CsA and with Ub0 by itself (Student's t test analysis; #: p<0.01), indicating that the inhibitory effect of both AA and CsA on the PTP is additive with that of Ub0.