Uncoupling of oxidative phosphorylation in rat liver mitochondria by general anesthetics.

Abstract

The general anesthetics chloroform and halothane inhibit ATP synthesis in rat liver mitochondria, in the millimolar concentration range (1-12 mM), in parallel with a reduction of respiratory control and the ratio of ATP produced to oxygen consumed. In these effects, halothane and chloroform are similar to classical, protonophoric, uncouplers. The rate of ADP-stimulated respiration or the rate of uncoupler-stimulated respiration is not affected. Like classical uncouplers, halothane and chloroform also stimulate mitochondrial ATPase activity. However, the extent of stimulation by these agents is larger than by protonophoric uncouplers and, more significantly, ATPase activity stimulated by carbonylcyanide m-chlorophenylhydrazone is further stimulated by these agents. In the presence of the Ca2+ chelator EGTA, halothane and chloroform have no measurable effect on the magnitude of the proton electrochemical potential, A!H. In the absence of EGTA these anesthetics have a small effect on AAH, apparently due to stimulation of Ca2+ cycling. Under these conditions the membrane potential is decreased while ApH is increased, but the total value of AIAH is only slightly decreased. The uncoupling activity of the anesthetics is the same in the presence or absence of EGTA. Thus, in contrast to protonophoric uncouplers, the uncoupling effect of general anesthetics does not depend on the collapse of AAH. In the same concentration range in which anesthetics uncouple oxidative phosphorylation both halothane and chloroform increase membrane fluidity, as measured by the partitioning of the hydrophobic spin probe 5-doxyldecane. These findings suggest a role for intramembrane processes in energy conversion that is not dependent on the bulkl AAH.Oxidative phosphorylation in mitochondria is believed to be mediated by the proton electrochemical potential gradient (ASH) as postulated by Mitchell in the chemiosmotic hypothesis (1-3). According to the chemiosmotic scheme, uncouplers are protonophores that catalyze the transport of protons across the membrane, thereby collapsing AAH, which is the direct driving force for ATP synthesis. The evidence for the protonophoric activity of uncouplers in both natural membranes and phospholipid bilayers is overwhelming (for review see ref. 4). In mitochondria, where ApH is relatively small, the collapse of membrane potential by electrophoretic transport of cations (e.g., Ca2" or K+ in the presence of valinomycin) is sufficient to decrease ASH and lead to uncoupling as well. Thus, to date, all known uncouplers of oxidative phosphorylation and photophosphorylation appear to collapse AAH in correlation with the inhibition of ATP synthesis (but see Discussion). However, several agents whose mechanism of action is still unknown are known to inhibit oxidative phosphorylation. Of particular interest are the general anesthetics, which are similar to protonophoric uncouplers in their effect on succinate respiration and on ATP synthesis and hydrolysis in mitochondria. Earlier studies, mostly with hal...

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