Enhanced uptake of spermidine and methylglyoxal-bis(guanylhydrazone) by rat liver mitochondria following outer membrane lysis (original) (raw)
Related papers
Biochemical Pharmacology, 1999
This study evaluated the effect of the anticancer drug methylglyoxal-bis(guanylhydrazone) (MGBG) on the binding of the polyamine spermine to the mitochondrial membrane and its transport into the inner compartment of this organelle. Spermine binding was studied by applying a new thermodynamic treatment of ligand-receptor interactions (Di Noto et al., Macromol Theory Simul 5: 165-181, 1996). Results showed that MGBG inhibited the binding of spermine to the site competent for the first step in polyamine transport; the interaction of spermine with this site, termed S 1 , also mediates the inhibitory effect of the polyamine on the mitochondrial permeability transition (Dalla Via et al., Biochim Biophys Acta 1284: 247-252, 1996. In the presence of 1 mM MGBG, the binding capacity and affinity of this site were reduced by about 2.6-fold; on the contrary, the binding capacity of the S 2 site, which is most likely responsible for the internalization of cytoplasmic proteins (see Dalla Via et al., reference cited above), increased by about 1.3-fold, and its binding affinity remained unaffected. MGBG also inhibited the initial rate of spermine transport in a dose-dependent manner by establishing apparently sigmoidal kinetics. Consequently, the total extent of spermine accumulation inside mitochondria was inhibited. This inhibition in transport seems to reflect a conformational change at the level of the channel protein constituting the polyamine transport system, rather than competitive inhibition at the inner active site of the channel, thereby excluding the possibility that the polyamine and drug use the same transport pathway. Furthermore, it is suggested that, in the presence of MGBG, the S 2 site is able to participate in residual spermine transport. MGBG also strongly inhibits ⌬pH-dependent spermine efflux, resulting in a complete block in the bidirectional flux of the polyamine and its sequestration inside the matrix space. The effects of MGBG on spermine accumulation are consistent with in vivo disruption of the regulator of energy metabolism and replication of the mitochondrial genome. membrane potential; and S 1 and S 2 , spermine binding sites 1 and 2.
Cancer research, 1980
The relationship between inhibition of polyamine biosyn thesis and interference with mitochondria! structure and func tion by the antitumor agent, methylglyoxal-bis(guanylhydrazone) (MGBG), was examined by studying the temporal sequence of events relevant to these two drug actions. In ascites L1210 cells treated in vivo with a single dose of MGBG (75 mg/kg), significant inhibition of pyruvate utilization, used here as a measure of mitochondrial function, occurred within 1 hr after initiation of drug treatment and continued to decrease for 24 hr. Ultrastructural damage to mitochondria, in the form of swelling, was first apparent in 50% of the cells after 6 hr of treatment and in > 90% of the cells after 20 hr. Inhibition of Sadenosylmethionine decarboxylase, a key enzyme in spermidine and spermine biosynthesis, occurred within 1 hr of drug treatment. Enzyme levels increased dramatically after 8 hr of treatment as a result of MGBG stabilization of the enzyme. Beginning at 8 hr, ornithine decarboxylase, the enzyme re sponsible for putrescine synthesis, increased slightly and con tinued to rise slowly during the next 16 hr. As a consequence of S-adenosylmethionine decarboxylase inhibition by MGBG, intracellular putrescine pools began to accumulate after 2 hr of treatment and increased rapidly after 12 hr. In contrast, sperm ine pools decreased slowly after 4 hr while spermidine pools decreased even more slowly. However, even after 24 hr, sig nificant amounts of both polyamines were still present intracellularly. Since MGBG-induced changes in mitochondrial function pre cede significant alterations in polyamine pools, it is concluded that the two effects are separable. This conclusion is further supported by the finding that, in cultured L1210 cells treated with a-methylornithine at concentrations which effectively in hibited putrescine and spermidine biosynthesis, the mitochon drial ultrastructure was unaffected. Results of the present study raise the possibility that MGBG interference with mitochondrial function might be responsible for the early antiproliferative action of the drug.
Actions of bis(guanylhydrazones) on isolated rat liver mitochondria
Biochemical Pharmacology, 1981
The effects of the anticancer bis(gu~ylhydrazones), methylglyoxal-bis(guanylhydrazone) (MGBG) and 4,4'-diacetyldiphenylurea-b~s(guanylhydrazone) @DUG), on parameters related to the bioenergetic function of isolated rat liver mitochondria were investigated, At concentrations comparable to those attained intracellularly, both bis(guanylhydrazones) si~ificantly inhibited state 4 respiration but had less of an inhibitor effect on state 3 or uncoupled respiration. DDUG was more effective than MGBG, requiring 0.34 mM to achieve a 25 per cent inhibition of respiration as compared to 6.0 mM for MGBG. The inhibition was prevented by potassium cations and was enhanced in mitochondria "de-energized" with valinomycin, a potassium cationophore. This suggested drug competition for potassium-binding sites, possibly membrane phospholipids. Addition of 1.25 mM MGBM or 0.025 mM DDUG to suspended mitochondria caused a rapid aggregation of organelles and an increase in the optical density of the suspension. Pretreatment with either bis(guanylhydrazone) protected mitochondria against non-specific swelling action of 0.0015% Triton X-100, suggesting membrane binding. By electron microscopy, MGBG-or DDUG-treated mitochondria appeared swollen and the spaces between cristae membranes or inner and outer membranes were collapsed, obliterating the outer mitochon~ial compartment. The activity of monoamine oxidase A, an outer membrane marker enzyme, was reduced considerably by 4 mM MGBG or 0.075 mM DDUG. Mobility of mitochondria toward the anode in an electrophoretic field was slowed 50 per cent by 2.5 mM MGBG or 0.1 mM DDUG, These data suggest that positively charged bis(~uanylhydr~ones) neutralize the net negative surface potential of rat liver mitochondria by binding to sites (possibly phospholipids) at the inner mitochondrial membrane. Subsequent interference with cation binding and/or transport results in inhibition of bioenergetic
Protective action of methylglyoxal bis (guanylhydrazone) on the mitochondrial membrane
Biochemical Pharmacology, 1988
At low concentrations (OS-l.0 mM) methylglyoxal bis (guanylhydrazone) (MGBG) exhibited a clearcut protection of rat liver mitochondria against the deenergizing action of either Ca*+, or oxidizing agents (butylhydroperoxide and oxaloacetate). Such a protection resulted from the prevention of transmembrane potential decay, discharge of accumulated Ca*+, release of mitochondrial Mg*+, adenine nucleotides and pyridine nucleotides and mitochondrial swelling. At high concentrations (S-10 mM) MGBG induced functional alterations of mitochondria (decrease of transmembrane potential, lower capability to accumulate and to retain CaL+) which can be reversed by resuspension of mitochondria in a MGBG free medium. These reversible mitochondrial alterations by high MGBG concentrations are interpreted as a consequence of an aggregation and coprecipitation of suspended mitochondria.
Inhibition of the bioenergetic functions of isolated rat liver mitochondria by polyamines
Biochemical Pharmacology, 1982
The abilities of the naturally occurring polyamines, putrescine, spermidine and spermine, to affect variables related to the bioenergetic functions of isolated rat liver mitochondria were studied. At concentrations comparable to those present intracellularly, the polyamines inhibited state 4 respiration, but they had much less effect on state 3 or uncoupled respiration. The concentrations required to produce 25% inhibition (I2s) of state 4 respiration varied according to the polyamine, with putrescine being least effective (125, 20 mM) and spermidine and spermine being more effective and comparable (I25, 7.5 and 7.0 mM respectively). This inhibition was antagonized by 15 mM potassium and enhanced by valinomycin and 4 mM magnesium. Inhibition of monoamine oxidase, an enzyme of outer mitochondrial membrane, was also observed to occur. Addition of polyamines to mitochondrial suspensions caused an increase in the optical density and protected against the swelling effects of sublytic concentrations of Triton X-100. By electron microscopy, polyamines were found to cause the outer mitochondrial compartment to collapse bringing the inner and outer membranes into apparent contact with one another. The electrophoretic mobility of mitochondria toward the anode was markedly slowed by polyamines (i.e. 50% by 1.25 mM spermine), indicating surface binding and neutralization of the negative surface charge. In almost all of the above mitochondrial effects, spermine and spermidine were similar in effectiveness and putrescine was less effective. It is suggested that polyamines may be capable of modulating respiration of isolated mitochondria by binding to non-specific anionic sites at the surface of the inner mitochondrial membrane. Neutralization of the net negative surface potential may interfere with cation fluxes across the membrane, particularly those of potassium.
Uptake of spermine by rat liver mitochondria and its influence on the transport of phosphate
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1985
Spermine, a polyamine present in the mammalian cells at rather high concentration, has, among other actions, a remarkable stabilizing effect on mitochondria, functions which have generally been attributed to the capability of this and other polyamines to bind to membrane anionic sites. In the present paper evidence is provided that at physiological concentrations spermine may also be transported into rat liver mitochondrial matrix space, provided that mitochondria are energized and inorganic phosphate is simultaneously transported. The close dependence of spermine transport is also demonstrated by the concurrent efflux of spermine and inorganic phosphate when mitochondria preloaded with the two ionic species are deenergized either with uncouplers or respiratory chain inhibitors. Furthermore, Mersalyl, the known inhibitor of phosphate transport, prevents both spermine uptake and release. Mg 2+ inhibits the transport of spermine conceivably by competing for the some binding sites on the mitochondrial membrane. The physiological significance of these results is discussed.
Binding of Spermidine and Putrescine to Energized Liver Mitochondria
Archives of Biochemistry and Biophysics, 1999
The binding of spermidine and putrescine to mitochondrial membranes was studied by applying a thermodynamic model of ligand-receptor interactions developed both for equilibrium and far-from-equilibrium binding processes (V. Di Noto, L. Dalla Via, A. Toninello, and M. Vidali Macromol. Theory Simul. 5, [165][166][167][168][169][170][171][172][173][174][175][176][177][178][179][180][181] 1996). Results demonstrate the presence of two monocoordinated binding sites (S 1 and S 2 ) for spermidine and one monocoordinated binding site (S 2 ) for putrescine, all exhibiting high capacity and low affinity. It is proposed that differences in the polyamines' flexibility and hydrophilicity perhaps contributes to the observed variations in their interactions with the two sites. A comparison of the binding parameters of these polyamines with those of spermine reveals differences in the specific function of the S 1 and S 2 sites, identified in studies of spermine binding (L.
Permeability of the mitochondrial outer membrane to organic cations
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1989
Inhibition of mitoc,~mS~al respiration by hydrophobic fluorescent dyes (Rhodamine 6G, Safranine O, Pyronine B) is much less potentiated by digitonin.iysis of the outer membrane than that by polyamines or adriamycin. This situation may be explained by ~mpenneability of the anion-selective channels in the outer mitochocdrial membrane to large cations and by ~qte abi.~ty of hydmp~F~ (but not polar or amphipathic) ions to directly permeate lipid bilayer~.
Free radical scavenging action of the natural polyamine spermine in rat liver mitochondria
Free Radical Biology and Medicine, 2006
The isoflavonoid genistein, the cyclic triterpene glycyrrhetinic acid, and salicylate induce mitochondrial swelling and loss of membrane potential (ΔΨ) in rat liver mitochondria (RLM). These effects are Ca 2+ -dependent and are prevented by cyclosporin A and bongkrekik acid, classic inhibitors of mitochondrial permeability transition (MPT). This membrane permeabilization is also inhibited by N-ethylmaleimide, butylhydroxytoluene, and mannitol. The above-mentioned pro-oxidants also induce an increase in O 2 consumption and H 2 O 2 generation and the oxidation of sulfhydryl groups, glutathione, and pyridine nucleotides. All these observations are indicative of the induction of MPT mediated by oxidative stress. At concentrations similar to those present in the cell, spermine can prevent swelling and ΔΨ collapse, that is, MPT induction. Spermine, by acting as a free radical scavenger, in the absence of Ca 2+ inhibits H 2 O 2 production and maintains glutathione and sulfhydryl groups at normal reduced level, so that the critical thiols responsible for pore opening are also consequently prevented from being oxidized. Spermine also protects RLM under conditions of accentuated thiol and glutathione oxidation, lipid peroxidation, and protein oxidation, suggesting that its action takes place by scavenging the hydroxyl radical.