Salicylates as Protonophores Uncoupling agents, such as 2,4-dinitrophenol or salicylate, increase the mitochondrial membrane proton conductance (Figure 2.22). This abolishes the energy-conserving proton gradient [211, 212] and results in bypassing the ATP synthase and release of protons into the matrix. In isolated mitochondria, the mito-chondrial membrane proton conductance is increased more than fourfold at 1 mM salicylate . Complete uncoupling occurs in model systems at 2-4 mM salicylate. The increasing H+/ Na+ exchange causes swelling of mitochondria [214, 215]. Swelling of mitochondria and reduced urea generation were also found in "primarily living" intact rat hepatocytes . This protono-phoric effect reduces the mitochondrial membrane potential that normally will activate the respiratory chain. The energy is now wasted in the form of heat, instead of generating ATP. Interestingly, there are marked differences in this activity between different salicylate-related hydro-xybenzoic acids .
At least two factors determine the activity of salicylate in uncoupling oxidative phosphorylation: the partition from an aqueous phase into a lipid-rich phase, allowing for penetration through the cell wall and access to the mitochondrion - the ultimate site of action. The second is a specific structural requirement to act as proto-
nophore. Structure-activity comparisons for uncoupling oxidative phosphorylation in isolated mitochondria of80 salicylate analogues showed that the essential pharmacophore for uncoupling activity is a salicylate with a negatively charged (carboxyl)group at the o-position, that is, o-hydroxybenzoate (salicylate) . The m- and p-hydroxybenzoate analogues were inactive. This suggests that the o-position of the hydroxyl group is an essential steric requirement for this protonophoric action .
Consequences of Uncoupling of Oxidative Phosphorylation by Salicylates Administration of high-dose aspirin to man causes a marked and progressive increase in oxygen consumption  because of uncoupling of oxidative phosphoryla-tion and becomes clinically evident as hyperventilation. This effect is dose dependent and typically occurs in initial stages of salicylate overdosing (Section 3.1.1) [221-224]. The uncoupling is not restricted to the liver but has also been found in isolated mitochondria of kidney, brain, and heart at higher salicylate concentrations (2-5 mM) [221, 225]. For thermodynamic reasons, glycogen-olysis and glycolysis are enhanced to provide the necessary ATP for cell functions. Uncoupling is a generally reversible process that can be terminated by removal of the agent and is then followed by complete recovery .
In rats, orally treated for up to 1 week with sodium salicylate at doses causing toxic side effects (hyperventilation, body wastage), there was no disturbed oxidative phosphorylation by isolated mitochondria ex vivo. In contrast, complete inhibition of mitochondrial oxidative phosphorylation was seen after in vitro treatment of isolated mitochondria with 5mM salicylate. The intracellular concentrations of salicylate in the liver of these rats after oral treatment were 0.8-4.0mM, that is, close to the plasma levels of salicylate. Salicylate was taken up and washed out from liver mitochondria within minutes, even at 0°C. The uncoupling, both in vitro and ex vivo, was reversible after washout of salicylate and did not cause irreversible tissue injury [212, 219].
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