Species-specific stimulation of the mitochondrial permeability transition by norbormide and its derivatives

Norbormide [5-(?-hydroxy-?-2-pyridylbenzyl)-7-(?-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB) is a toxic compound endowed with a unique species-specific vasoconstrictor activity that is restricted to the peripheral arteries of the rat. It is a mixture of eight racemate diastereoisomers, which strongly differ in their vasoconstrictor activity1. Early studies demonstrated that NRB also causes rat-specific mitochondrial dysfunction, which was recently attributed to the opening of the inner membrane permeability transition pore (PTP)2. A question posed by these findings was whether the species-specific PTP modulation and the species-specific toxicity are causally linked, as suggested by the fact that both processes are specific to the rat. To address this question, in the present study we investigated the PTP-regulatory properties of both vasoconstrictor and non vasoconstrictor NRB isomers by following the Ca2+ retention capacity and matrix swelling of rat, mouse and guinea pig liver mitochondria. All NRB isomers tested exihibited similar, rat-specific effects on mitochondrial permeabilization, which indicates that: i) species-specifity of NRB action on the PTP is not connected to the molecular isomerism; ii) regulation of pore activity and vasoconstrictor activity are unrelated phenomena. Our previous work2 showed that NRB induced rat-specific changes in the fluidity of mitochondrial membranes, which in turn were likely to increase the PTP open probability through conformational changes of the internal Ca2+ regulatory site. Based on these observations, the mechanism of NRB action on the PTP was supposed to involve a mitochondrial transport system unique to the rat that allows internalization of the drug across the membranes. This putative carrier should be absent in other species, or shielded from contact with the drug due to different membrane structural arrangements2. In order to better understand the mechanism of NRB action, we carried out studies with cationic derivatives of the drug, which are accumulated inside the mitochondria via the transmembrane potential of the inner membrane. Cationic NRB stimulated the opening of the PTP at much lower concentrations than the neutral molecule (2 ?M as compared to 35 ?M in rat mitochondria) and was effective also on mouse and guinea pig mitochondria. The loss of species-specificity on the PTP opening by the NRB cationic derivative is consistent with our previous hypothesis that the neutral drug is taken up by rat mitochondria via a selective carrier.