Effects of Guanidine Derivatives on Mitochondrial Function: Ca2+ UPTAKE AND RELEASE

Abstract

Abstract Succinate-energized guinea pig heart mitochondria prepared by the Polytron technique take up Ca2+ under limited loading conditions in a manner similar to mitochondria prepared with bacterial proteinase, despite the associated loss of high affinity nonenergized Ca2+ binding by proteinase-prepared mitochondria. Phenethylbiguanide inhibits initial energized Ca2+ uptake; 50 % inhibition occurs at 2 mm biguanide in both Polytron and proteinase preparations and this inhibition is at least partly dissociated from inhibition of substrate oxidation. Energized heart mitochondria lose accumulated Ca2+ by a process dependent on temperature, Ca2+ to protein ratio, and monovalent cation. Phenethylbiguanide also inhibits this Ca2+ release. Nonenergized Ca2+ binding is 50 % inhibited by 1.0 mm biguanide, and inhibition is intermediate between competitive and non-competitive. Phenethylbiguanide also inhibits Ca2+ uptake into both energized and nonenergized guinea pig liver mitochondria; 50% inhibition occurs at 1.1 and 8.5 mm biguanide, respectively. In contrast to heart mitochondria, biguanide inhibition of energized Ca2+ uptake into liver mitochondria exactly parallels inhibition of Ca2+ and of ADP + Pi-stimulated respiration. Biguanide inhibition of nonenergized Ca2+ binding to liver mitochondria is competitive. [14C]Phenethylbiguanide is taken up by energized and nonenergized heart and liver mitochondria; binding capacity is about 60 nmoles per mg of protein. At 1 mm biguanide, nonenergized heart mitochondria bind about 6 nmoles per mg of protein; energized heart mitochondria accumulate labeled biguanide in a rapid phase, which corresponds to inhibition of initial 45Ca2+ uptake, and a slower, progressive uptake phase. The data suggest that both energized Ca2+ movement and biguanide binding occur at the locus which accounts for low affinity Ca2+ binding in the absence of energy. Site-specific inhibition of coupled respiration may result from biguanide binding primarily in or near this low affinity Ca2+ locus. The high affinity locus may play a modulator role in energized Ca2+ transport and in respiratory inhibition by biguanides. Interference with mitochondrial Ca2+ transport may in part explain the cellular effects of guanidine derivatives.