Rhodamine 6G: A POTENT INHIBITOR OF MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION

Abstract

Abstract The lipophilic dye, rhodamine 6G, has been shown to be a potent inhibitor of oxidative phosphorylation, with a Ki of about 3 µm, corresponding to 1.2 nmoles of dye per mg of mitochondrial protein. ATP-supported Ca2+ accumulation was blocked, but not that driven by succinate oxidation. Concentrations of rhodamine 6G above 20 µm slowly uncouple respiration and inhibit respiration-dependent Ca2+ uptake. Neither the dinitrophenol-stimulated ATPase nor uncoupled respiration of intact mitochondria were inhibited by the dye, even at 50 µm. Arsenate-stimulated respiration, on the other hand, was partially blocked by 7.5 µm dye, but energy-linked phosphate accumulation was unaffected. Below 10 µm rhodamine 6G, no mitochondrial swelling, loss of matrix protein, or endogenous K+, Ca2+, or Mg2+, was observed. The dye was bound very tightly to mitochondria, being present at 1.3 and 0.8 nmoles per mg of protein for the inner and outer membranes, respectively. Binding was also monitored by direct measurement of H+ release into the suspending medium in the presence of rhodamine 6G. Some 0.6 to 0.75 mole of H+ was ejected per mole of dye added, of which only about 50% was firmly bound. A red shift occurred on binding, the λmax increasing from 527 to 535 nm. The related compound, rhodamine B, a free acid and uncharged at pH 7, was completely without influence on mitochondrial energy-linked functions. Kinetic studies on the rate of ADP-stimulated H+ uptake in the presence and absence of rhodamine 6G yielded nonlinear, Lineweaver-Burk plots and were of noncompetitive type. The Km for ADP decreased from 56 to 29 µm in the presence of 4 µm rhodamine 6G. When the data is expressed as a Hill plot, straight lines are obtained, with the value of (η), the interaction coefficient, increasing from 1.28 to 1.72 after dye addition. Rhodamine 6G did not inhibit the Mg2+ATPase in sonicated submitochondrial particles, unlike aurovertin. It did, however, block adenine nucleotide binding to intact mitochondria, both for [14C]ATP and [14C]ADP. Inhibition by 10 µm dye for low levels of added nucleotide was identical with that caused by 10 µm atractyloside, but was only very slight at higher nucleotide concentrations (0.2 mm). The results of this study support the conclusion that rhodamine 6G blocks the adenine nucleotide translocase apparently being similar to atractyloside and bongkrekic acid. However, in sharp contrast to these inhibitors, rhodamine 6G did not inhibit the 2,4-dinitrophenol-stimulated ATPase of intact mitochondria. Consequently, uncouplers destroy the dye's ability to interfere with the translocase, and this suggests a lipid binding to be involved in the action of rhodamine 6G.