Direct evidence for the control of mitochondrial respiration by mitochondrial creatine kinase in oxidative muscle cells in situ

The efficiency of stimulation of mitochondrial respiration in permeabilized muscle cells by ADP produced at different intracellular sites, e.g cytosol ic or mitochondrial intermembrane space, was evaluated in wildtype and crea tine kinase (CK)-deficient mice. To activate respiration by endogenous prod uction of ADP in permeabilized cells, ATP was added either alone or togethe r with creatine, In cardiac fibers, while ATP alone activated respiration t o half of the maximal rate, creatine plus ATP increased the respiratory rat e up to its maximum. To find out whether the stimulation by creatine is a c onsequence of extramitochondrial [ADP] increase, or whether it directly cor relates with ADP generation by mitochondrial CK in the mitochondrial interm embrane space, an exogenous ADP-trap system was added to rephosphorylate al l cytosolic ADP, Under these conditions, creatine plus ATP still increased the respiration rate by 2.5 times, compared with ATP alone, for the same ex tramitochondrial [ADP] of 14 mu M, Moreover, this stimulatory effect of cre atine, observed in wild-type cardiac fibers disappeared in mitochondrial CK deficient, but not in cytosolic CK-deficient muscle. It is concluded that respiration rates can be dissociated from cytosolic [ADP], and ADP generate d by mitochondrial CK is an important regulator of oxidative phosphorylatio n.