Polyamine modulation of mitochondrial calcium transport. II. Inhibition of mitochondrial permeability transition by aliphatic polyamines but not by aminoglucosides.

In this study, the effects of polyamines and analogous compounds on mitochondrial permeability transition were characterized to distinguish between these effects and those on mitochondrial Ca2+ uptake, which are described in an accompanying report (Rustenbeck et al., Biochem Pharmacol 8: 977-985, 1998). When a transitional Ca2+ release from Ca2+-loaded mitochondria was induced by an acute increase in Ca2+ concentration in a cytosol-adapted incubation medium (Ca2+ pulse), this process was inhibited, but not abolished by spermine in the concentration range of 0.4 to 20 mM. The aminoglucoside, gentamicin, and the basic polypeptide, poly-L-lysine, which like spermine are able to enhance mitochondrial Ca2+ accumulation (preceding paper), had no or only a minimal inhibitory effect, while the aliphatic polyamine, bis(hexamethylene)triamine, which is unable to enhance mitochondrial Ca2+ accumulation, achieved a complete inhibition at 4 mM. The conclusion that the Ca2+ efflux was due to opening of the permeability transition pore was supported by measurements of mitochondrial membrane potential, ATP production, and oxygen consumption. Mg2+, a known inhibitor of mitochondrial membrane permeability transition, did not mimic the effects of spermine on mitochondrial Ca2+ accumulation, while ADP, the main endogenous inhibitor, showed both effects. However, a combination of spermine and ADP was significantly more effective than ADP alone in restoring low Ca2+ concentrations after a Ca2+ pulse. Two different groups of spermine binding sites were found at intact liver mitochondria, characterized by dissociation constants of 0.5 or 4.7 mM and maximal binding capacities of 4.6 or 19.7 nmol/mg of protein, respectively. In contrast to aminoglucosides, the aliphatic polyamine bis(hexamethylene)triamine did not displace spermine from mitochondrial binding sites. The total intracellular concentration of spermine in hepatocytes was measured to be ca. 450 microM and the free cytoplasmic concentration was estimated to be in the range of 10-100 microM. In conclusion, the enhancement of mitochondrial Ca2+ uptake by spermine is not an epiphenomenon of the inhibition of permeability transition. The physiological role of spermine appears to be that of an enhancer of mitochondrial Ca2+ accumulation rather than an inhibitor of permeability transition.

Degradation of dansyl polyamines on high-performance thin-layer chromatographic plates.

Using high-performance thin-layer chromatography with in situ quantitation to measure dansylated polyamines in the range of 1-20 pmol, we found that dansylated polyamines apparently react with the silica gel of the plates. The fluorescence of the dansyl polyamines diminished with increase in the time interval between application of a sample to the plate and start of the chromatographic separation. Conversely, the fluorescence at the site of application increased with the length of the time interval, indicating the formation of polar reaction products. If this reaction is not accounted for, considerable errors in quantitation of dansyl polyamines may occur.