mersalyl and oxophenylarsine

The purpose of this work was addressed to provide new information on the effect of thiol reagents on mitochondrial non-specific pore opening, and its response to cyclosporin A (CSA). To meet this proposal phenylarsine oxide (PHA) and mersalyl were employed as tools to induce permeability transition and CSA to inhibit it. PHA-induced mitochondrial dysfunction, characterized by Ca2+ efflux, swelling, and membrane de-energization, was inhibited by N-ethylmaleimide and CSA. Conversely, mersalyl failed to inhibit the inducing effect of phenylarsine oxide, it rather strengthened it. In addition, the effect of mersalyl was associated with cross-linking of membrane proteins. The content of membrane thiol groups accessible to react with PHA, mersalyl, and PHA plus mersalyl was determined. In all situations, permeability transition was accompanied by a significant decrease in the whole free membrane thiol content. Interestingly, it is also shown that mersalyl hinders the protective effect of cyclosporin A on PHA-induced matrix Ca2+ efflux.

Topics: Animals; Arsenicals; Calcium; Cross-Linking Reagents; Cyclosporine; Ethylmaleimide; Ion Channel Gating; Membrane Potential, Mitochondrial; Mersalyl; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Mitochondrial Membranes; Mitochondrial Swelling; Permeability; Porins; Protein Binding; Rats; Sulfhydryl Compounds; Sulfhydryl Reagents

The content of mitochondrial membrane protein thiol groups accessible to react with the monofunctional thiol reagents mersalyl or N-ethylmaleimide (NEM) was determined using Ellman's reagent. Deenergized mitochondria incubated in the presence of Ca2+ (0-500 microM) undergo a very significant decrease in the content of membrane protein thiols accessible to NEM, and an increase in the content of thiols accessible to mersalyl. This process is time-dependent and inhibited by Mg2+, ruthenium red and ADP, but not by cyclosporin A. This suggests that Ca2+ binding to the inner mitochondrial membrane promotes extensive alterations in the conformation of membrane proteins that result in location changes of thiol groups. The relationship between these alterations and mitochondrial membrane permeability transition was studied through the effect of NEM and mersalyl on mitochondrial swelling induced by Ca2+ plus t-butyl hydroperoxide (t-bOOH) or Ca2+ plus the thiol cross-linkers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or phenylarsine oxide (PhAsO). We observed that the hydrophobic thiol reagent NEM inhibits the effects of t-bOOH, DIDS and PhAsO, while the hydrophilic thiol reagent mersalyl inhibits only the effect of DIDS. Permeability transition in all the situations studied is accompanied by a significant decrease in the total membrane protein thiol content. In addition, mitochondrial membrane permeabilization induced by PhAsO is inhibited by EGTA, but not by ruthenium red. This result suggests that PhAsO leads to permeability transition through a mechanism independent of intramitochondrial Ca2(+)-induced alterations of thiol group reactivity, but dependent on Ca2+ binding to an extramitochondrial site. This site is sensitive to extramitochondrial Ca2+ concentrations in range of 1-50 microM.

Topics: Animals; Arsenicals; Binding Sites; Calcium; Ethylmaleimide; In Vitro Techniques; Intracellular Membranes; Membrane Proteins; Mersalyl; Mitochondria, Liver; Mitochondrial Swelling; Permeability; Peroxides; Rats; Rats, Wistar; Sulfhydryl Compounds; Sulfhydryl Reagents; tert-Butylhydroperoxide

The effect of the conformational state of the ADP/ATP-antiporter on the efficiency of inhibition by cyclosporine A of the Ca(2+)-induced increase of nonspecific permeability of the inner mitochondrial membrane was under study. It was found that the ADP/ATP-antiporter inhibitor carboxyatractyloside is able to reverse the cyclosporine A-induced inhibition of this nonspecific permeability. The effect of carbocyatractyloside is manifested only in mitochondria depleted of adenine nucleotides. The bifunctional SH-reagent phenylarsine oxide is also able to reverse the cyclosporine A effect. The data obtained testify to the fact that inhibition by cyclosporine A of nonspecific permeability is due to its effect on the conformational state of the ADP/ATP-antiporter.

Topics: Arsenicals; Calcium; Cyclosporins; Ethylmaleimide; Intracellular Membranes; Mersalyl; Mitochondria, Liver; Mitochondrial ADP, ATP Translocases; Permeability

The dithiol-reactive reagent phenylarsine oxide causes a pH-dependent stimulation of unidirectional K+ flux into respiring rat liver mitochondria. This stimulation is diminished by subsequent addition of either the dithiol 2,3-dimercaptopropanol or the monothiol 2-mercaptoethanol. In contrast, uncoupling by phenylarsine oxide is reversed by 2,3-dimercaptopropanol but not by 2-mercaptoethanol. The data suggest separate sites of interaction of phenylarsine oxide with mechanisms of K+ entry and ATP synthesis. Stimulatory effects of mersalyl and phenylarsine oxide on K+ influx are not additive. Thus PheASO and mersalyl may affect K+ influx at a common site. Pretreatment of the mitochondria with DCCD, which inhibits K+ influx, fails to alter sensitivity to PheAsO or mersalyl. Thus the DCCD binding site associated with the K+ influx mechanism appears to be separate from and independent of the sulfhydryl group(s) which mediate stimulation of K+ influx by PheAsO and mersalyl. PheAsO, like mersalyl, also increases the rate of unidirectional K+ efflux from respiring mitochondria. The combined presence of PheAsO plus mersalyl causes a greater stimulation of K+ efflux than is observed with either reagent alone.

Topics: Animals; Arsenicals; Biological Transport, Active; Dicyclohexylcarbodiimide; Kinetics; Mercaptoethanol; Mersalyl; Mitochondria, Liver; Oxygen Consumption; Potassium; Rats