INHIBITION OF MEMBRANE LIPID-PEROXIDATION BY A RADICAL SCAVENGING MECHANISM - A NOVEL FUNCTION FOR HYDROXYL-CONTAINING IONOPHORES

In the present study we show that K+/H+ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe2+-citrate and 2,2'-azobis(2-amidinopropane) dihydroc hloride (ABAP)-induced lipid peroxidation in intact rat liver mitochon dria and in egg phosphatidylcholine (PC) liposomes containing negative ly charged lipids-dicetyl phosphate (DCP) or cardiolipin (CL)- and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-conta ining ionophore with higher affinity for Na+ than for K+, promotes a s imilar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-inse nsitive O-2 consumption, formation of thiobarbituric acid-reactive sub stances (TBARS), conjugated dienes, and electron paramagnetic resonanc e (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid p eroxidation induced by both inductor systems in liposomes. Nitroxide d estruction is inhibited by butylated hydroxytoluene, a known antioxida nt, and by the hydroxyl-containing ionophores. In contrast, valinomyci n (VAL), which does not possess alcoholic groups, does not display thi s protective effect. Effective order parameters (S-eff), determined fr om the spectra of an incorporated spin label are larger in the presenc e of salt and display a small increase upon addition of the ionophores , as a result of the increase of counter ion concentration at the nega tively charged bilayer surface. This condition leads to increased form ation of the ion-ionophore complex, the membrane binding (uncharged) s pecies. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution ti n the absence of membranes) showed that NIG and LAS, but not VAL, decr ease the Fe2+-citrate-induced production of radicals derived from pipe razine-based buffers, demonstrating their property as radical scavenge rs. Both Fe2+-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatid ylcholine (DMPC, saturated phospholipid)-DCP Liposomes, indicating tha t the ionophore also scavenges Lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all Lipid compo sitions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack th e ionophore. Altogether, the data lead to the conclusion that the memb rane-incorporated cation complexes of NIG, LAS and MON inhibit lipid p eroxidation by blocking initiation and propagation reactions in the li pid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and t o the attack of the aromatic moiety of LAS.