Mechanisms of Inhibition and Uncoupling of Respiration in Isolated Rat Liver Mitochondria by the General Anesthetic 2,6‐Diisopropylphenol

Abstract

We investigated the effects of 2,6-diisopropylphenoI on oxidative phosphorylation of isolated rat liver mitochondria. Diisopropylphenol strongly inhibits state-3 and uncoupled respiratory rates, when glutamate and malate are the substrates, as a direct consequence of the limitation of electron transfer at the level of complex I. In addition, diisopropylphenol acts as an uncoupler in non-phosphorylating mitochondria, which leads to an increase in respiratory rate and a large decrease in proton-motive force. However, such effects cannot be due to the classical protonophoric property of this drug, since addition of ADP plus oligomycin before diisopropylphenol avoids this increase in proton permeability, and in phosphorylating mitochondria, the ATP/O ratio is not significantly affected by diisopropylphenol addition. In the absence of added ADP, diisopropylphenol modifies some mitochondrial ATPases in such a way that they become insensitive to oligomycin and unable to couple proton movement to ATP synthesis or hydrolysis. However. these modified enzymes can catalyse passive proton permeability, which leads to uncoupling. Addition of ADP before diisopropylphenol prevents these changes. We propose that ADP induces a change in conformation of ATPase, which leads to insensitivity of this complex towards diisopropylphenol.In conclusion, we show that diisopropylphenol has two main effects on rat liver mitochondria: inhibition of the respiratory chain at the level of complex I level and modification of ATPase such that, in the absence of phosphorylation, it catalyses a H' leak, which becomes negligible when oxidative phosphorylation is functional.Keywords: 2,6-diisopropylphenoI; rat liver mitochondria; respiratory chain ; oxidative phosphorylation ; adenosine triphosphatase.Even though general anesthetics have a great variety of molecular structures and have been reported to affect different mitochondrial functions, most have been characteri~ed as uncouplers of oxidative phosphorylation. For instance, halothane and chloroform induce stimulation of state-4 respiration, inhibition of ATP synthesis and increase basal ATPase activity; all these effects define an uncoupling process of oxidative phosphorylation [I -31. However, in contrast to the wide class of classical uncouplcrs, c.g. protonophores or ionophores, halothane and chloroform only very slightly increase the proton and/or ion conductance of the inner mitochondrial membrane and cause only a slight depression of proton-motive force (LIP). That these strong uncouplers can act without a significant effect on bulk-to-bulk d p has been considered as evidence in favour of the existence of either a parallel (intramembranal) coupling pathway for pro- H ' -transporting ATP synthase (EC 3.6.1 34).tons [4] or a mechanism of intrinsic uncoupling at the protonpump level 151.2,6-diisopropyIphenol, an alkyl-substituted phenol currently used as an intravenous general anesthetic, has previously been reported to reduce the transmembrane difference in electrical potential and to increas...

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