Protective Effects of Some Creatine Derivatives in Brain Tissue Anoxia

Some derivatives more lipophylic than creatine, thus theoretically being capable to better cross the blood–brain barrier, were studied for their protective effect in mouse hippocampal slices. We found that N-amidino-piperidine is harmful to brain tissue, and that phosphocreatine is ineffective. Creatine, creatine–Mg-complex (acetate) and phosphocreatine–Mg-complex (acetate) increased the latency to population spike disappearance during anoxia. Creatine and creatine–Mg-complex (acetate) also increased the latency of anoxic depolarization, while the delay induced by phosphocreatine–Mg-complex (acetate) was of borderline significance (P = 0.056). Phosphocreatine–Mg-complex (acetate) significantly reduced neuronal hyperexcitability during anoxia, an effect that no other compound (including creatine itself) showed. For all parameters except reduced hyperexcitability the effects statistically correlated with tissue levels of creatine or phosphocreatine. Summing up, exogenous phosphocreatine and N-amidino piperidine are not useful for brain protection, while chelates of both creatine and phosphocreatine do replicate some of the known protective effects of creatine. In addition, phosphocreatine–Mg-complex (acetate) also reduced neuronal hyperexcitability during anoxia.     

Role of creatine and phosphocreatine in neuronal protection from anoxic and ischemic damage

Summary.  Phosphocreatine can to some extent compensate for the lack of ATP synthesis that is caused in the brain by deprivation of oxygen or glucose. Treatment of in vitro rat hippocampal slices with creatine increases the neuronal store of phosphocreatine. In this way it increases the resistance of the tissue to anoxic or ischemic damage. In in vitro brain slices pretreatment with creatine delays anoxic depolarization (AD) and prevents the irreversible loss of evoked potentials that is caused by transient anoxia, although it seems so far not to be active against milder, not AD-mediated, damage. Although creatine crosses poorly the blood-brain barrier, its administration in vivo at high doses through the intracerebroventricular or the intraperitoneal way causes an increase of cerebral phosphocreatine that has been shown to be of therapeutic value in vitro. Accordingly, preliminary data show that creatine pretreatment decreases ischemic damage in vivo. Received July 3, 2001 Accepted August 6, 2001 Published online July 31, 2002     

Preincubation with Creatine Enhances Levels of Creatine Phosphate and Prevents Anoxic Damage in Rat Hippocampal Slices

Abstract: We have investigated the relationship between energy metabolism, NMDA-receptor antagonism, and anoxic damage in vitro. Anoxic damage was assessed by measuring protein synthesis, defined as the incorporation of [¹⁴C]lysine into perchloric acid-insoluble tissue extracts. The concentrations of energy metabolites were measured by ion-exchange HPLC. Anoxia caused an inhibition of protein synthesis, a reduction in phosphocreatine and adenosine triphosphate, and extensive neuronal damage. The reduction of protein synthesis depended on the duration of anoxia and the time allowed for recovery. Preincubation with the creatine dose-dependently (0.03–3 mmol/L) increased baseline levels of phosphocreatine, reduced the anoxia-induced decline in phosphocreatine and adenosine triphosphate, prevented the impairment of protein synthesis, and reduced neuronal death. Incubation with ( R,S )-3-guanidinobutyric acid, a synthetic analogue of creatine that cannot be phosphorylated, did not prevent the anoxia-induced impairment of protein synthesis and did not enhance the levels of phosphocreatine and adenosine triphosphate. Incubation with a combination of both creatine and the noncompetitive NMDA antagonist MK-801 provided complete protection. These results indicate that energy status is a major factor controlling anoxic damage in the rat hippocampal slice.     

Cerebral Synaptic Transmission During Anoxia Is Protected by Creatine

Synaptic transmission in cerebral tissue fails very rapidly in the absence of oxygen; the metabolic basis for this is not known. We report here that the transmission failure in the guinea pig hippocampal slice can be delayed threefold by exposing the tissue to extracellular creatine (Cr) for 3 h. The improved survival is associated with an increase of tissue phosphocreatine (PCr) concentration. These data argue that the metabolic basis for synaptic transmission failure is a fall in tissue ATP concentrations. They also indicate a way to protect brain tissue against anoxic damage.     

Ability of a phosphocreatine-myofibrillar creatine kinase system to prevent the rigor tension of myocardial fibers

In the calcium-free medium the EGTA-treated rat myocardial fibres developed rigor tension dependent on the concentration of MgATP in the bathing solution: half-maximal tension was recorded at 2.5 mM MgATP and the maximal tension at 0.1 mM. However, in the presence of 15 mM phosphocreatine without added creatine kinase a decrease of MgATP concentration to 0.1 mM did not result in any development of rigor tension. In the presence of MgADP phosphocreatine decreased rigor tension more rapidly and to the higher extent than MgATP. At 5 mM MgADP half-maximal rigor tension was observed in the presence of 2 mM phosphocreatine which is close to the km value for phosphocreatine in the creatine kinase reaction. These results demonstrate that the native creatine kinase in the EGTA-treated fibres is able to create high local ATP concentration in the myofibrillar compartment at the expense of phosphocreatine under the conditions of deficiency or even absence of ATP. It appears that at the energy supply disturbances the myocardial contracture develops at least partially due to low activity of the myofibrillar creatine kinase because of phosphocreatine deficiency.     

Creatine transporter localization in developing and adult retina: importance of creatine to retinal function

Creatine and phosphocreatine are required to maintain ATP needed for normal retinal function and development. The aim of the present study was to determine the distribution of the creatine transporter (CRT) to gain insight to how creatine is transported into the retina. An affinity-purified antibody raised against the CRT was applied to adult vertebrate retinas and to mouse retina during development. Confocal microscopy was used to identify the localization pattern as well as co-localization patterns with a range of retinal neurochemical markers. Strong labeling of the CRT was seen in the photoreceptor inner segments in all species studied and labeling of a variety of inner neuronal cells (amacrine, bipolar, and ganglion cells), the retinal nerve fibers and sites of creatine transport into the retina (retinal pigment epithelium, inner retinal blood vessels, and perivascular astrocytes). The CRT was not expressed in Müller cells of any of the species studied. The lack of labeling of Müller cells suggests that neurons are independent of this glial cell in accumulating creatine. During mouse retinal development, expression of the CRT progressively increased throughout the retina until approximately postnatal day 10, with a subsequent decrease. Comparison of the distribution patterns of the CRT in vascular and avascular vertebrate retinas and studies of the mouse retina during development indicate that creatine and phosphocreatine are important for ATP homeostasis. photoreceptor; development; glutamine synthetase; neurochemistry     

Creatine deficiency syndromes and the importance of creatine synthesis in the brain

Creatine deficiency syndromes, due to deficiencies in AGAT, GAMT (creatine synthesis pathway) or SLC6A8 (creatine transporter), lead to complete absence or very strong decrease of creatine in CNS as measured by magnetic resonance spectroscopy. Brain is the main organ affected in creatine-deficient patients, who show severe neurodevelopmental delay and present neurological symptoms in early infancy. AGAT- and GAMT-deficient patients can be treated by oral creatine supplementation which improves their neurological status, while this treatment is inefficient on SLC6A8-deficient patients. While it has long been thought that most, if not all, of brain creatine was of peripheral origin, the past years have brought evidence that creatine can cross blood–brain barrier, however, only with poor efficiency, and that CNS must ensure parts of its creatine needs by its own endogenous synthesis. Moreover, we showed very recently that in many brain structures, including cortex and basal ganglia, AGAT and GAMT, while found in every brain cell types, are not co-expressed but are rather expressed in a dissociated way. This suggests that to allow creatine synthesis in these structures, guanidinoacetate must be transported from AGAT- to GAMT-expressing cells, most probably through SLC6A8. This new understanding of creatine metabolism and transport in CNS will not only allow a better comprehension of brain consequences of creatine deficiency syndromes, but will also contribute to better decipher creatine roles in CNS, not only in energy as ATP regeneration and buffering, but also in its recently suggested functions as neurotransmitter or osmolyte.     

Role of phosphocreatine in energy transport in skeletal muscle of bullfrog studied by 31P-NMR

To evaluate the energy-shuttle hypothesis of the phosphocreatine/creatine kinase system, diffusion rates for ATP, phosphocreatine and flux through the creatine kinase reaction were determined by 31P-NMR in resting bullfrog biceps muscle. The diffusion coefficient of phosphocreatine measured by 31P-pulsed gradient NMR was 1.4-times larger than ATP in the muscle, indicating the advantage of phosphocreatine molecules for the intracellular energy transport. The flux of the creatine kinase reaction measured by 31P-saturation transfer NMR was 3.6 mmol/kg wet wt. per s in the resting muscle. The flux is equal to the turnover rate of ATP, ADP, phosphocreatine and creatine molecules, therefore, the life-times of these substrates and the average distance traversed after the life-times by the diffusing molecules were calculated using the diffusion coefficients obtained by 31P-NMR. The mean square length of one-dimensional diffusion was 22 microns in ATP molecules and the minimum diffusion length was 1.8 microns in ADP molecules. The latter was calculated using free ADP concentration, 30 mumol/kg wet wt., obtained from the equilibrium constant of the creatine kinase reaction and the diffusion coefficient assumed to be the same of ATP in muscle. Similar diffusion lengths of ADP were calculated using the reported values for the flux of the creatine kinase reaction in heart and smooth-muscle. The diffusion lengths of all substrates involved in the creatine kinase reaction were larger than the radii of myofibrils. Therefore, in the muscles with an alternating arrangement of mitochondria and myofibrils, such as heart and certain skeletal muscles, ATP and ADP molecules can move freely between myofibrils and mitochondria without the aid of the creatine kinase reaction; thus, we conclude that the energy-shuttle hypothesis is not obligatory for energy transport between the mitochondria and the myofibrils.     

Creatine supplementation affects sprint endurance in juvenile rainbow trout

Fingerling rainbow trout were supplemented with equal amounts of creatine (Cr) by two routes: dietary (12.5 mg Cr per g food); or intraperitoneal injection (0.5 mg Cr per g fish). Endurance in a fixed velocity sprint test (at a speed of 7 BL s(-1)), and resting levels of white muscle metabolites (total creatine [a measure of free creatine plus phosphocreatine (PCr), ATP, lactate and glycogen] were assessed following 7 days of supplementation and compared to controls. None of the treatments had a significant effect on growth, muscle total creatine, percent phosphorylation of creatine, ATP or lactate. However, resting muscle glycogen was elevated in creatine-supplemented fish. Higher muscle glycogen corresponded to significantly greater endurance in creatine-supplemented fish. Although fish do not actively transport additional creatine into the muscle, a mechanism whereby circulating creatine acts to enhance muscle glycogen is present. These results suggest that the improved endurance may be due to an insulin-dependent mechanism (similar to that elucidated in mammalian studies) that allows fish to supercompensate muscle glycogen stores, thus extending endurance through enhanced glycolytic flux.     

Phosphoinositide Breakdown in Rat Hippocampal Slices: Sensitivity to Glutamate Induced by In Vitro Anoxia

We examined the effects of in vitro anoxia on phosphoinositide (PI) breakdown in rat hippocampal slices stimulated by glutamate and quisqualate. In addition to assays of accumulations of 3H-inositol phosphates (3H-IPs) degraded from prelabeled PI, we adopted direct assay procedures of inositol 1,4,5-triphosphate (1,4,5-IP3) using 1,4,5-IP3-specific binding protein to determine the formation of 1,4,5-IP3. The first effect, observed with anoxic incubation by itself, was the diminished quisqualate (10(-5) M)-stimulated accumulation of 3H-IPs degraded from prelabeled PI under prolonged anoxia. Quisqualate caused a transient increase in 1,4,5-IP3 formation in the early phase of anoxia, similar to that under oxygenated conditions. Glutamate (10(-5) M), under normal conditions, influenced neither the accumulation of 3H-IPs nor the formation of 1,4,5-IP3. Also, the accumulation of 3H-IPs under prolonged anoxia was unaffected. The same concentration of glutamate, however, gave rise to a transient increase in 1,4,5-IP3 content in the early phase of anoxia, similar to that caused by quisqualate. The second effect, observed by oxygenation following anoxia, was the induction of glutamate-stimulated accumulation of 3H-IPs. When the hippocampal slices were oxygenated following a sufficiently long (greater than 30-min) exposure to anoxia, glutamate (10(-5) M) caused a significant increase in accumulation of 3H-IPs degraded from prelabeled PI. Quisqualate-stimulated accumulation of 3H-IPs under oxygenated incubations was also increased by prior exposure of slices to anoxia. These results support the hypothesis that an exposure of hippocampal slices to anoxia induces a sensitivity of the PI breakdown pathway to glutamate and that, given an oxygen supply following sufficiently long exposure to anoxia, the slices maintain their sensitivity to glutamate with an apparent increase in the accumulation of 3H-IPs.     

Creatine kinase of heart mitochondria. Control of oxidative phosphorylation by the extramitochondrial concentrations of creatine and phosphocreatine

Defining how extramitochondrial high-energy phosphate acceptors influence the rates of heart oxidative phosphorylation is essential for understanding the control of myocardial respiration. When the production of phosphocreatine is coupled to electron transport via mitochondrial creatine kinase, the net reaction can be expressed by the balanced equation: creatine + Pi----phosphocreatine + H2O. This suggests that rates of oxygen consumption could be regulated by changes in [creatine], [Pi], or [phosphocreatine], alone or in combination. The effects of altering these metabolites upon mitochondrial rates of respiration were examined in vitro. Rat heart mitochondria were incubated in succinate-containing oxygraph medium (pH 7.2, 37 degrees C) supplemented with five combinations of creatine (1.0-20 mM), phosphocreatine (0-25 mM), and Pi (0.25-5.0 mM). In all cases, the mitochondrial creatine kinase reaction was initiated by additions of 0.5 mM ATP. To emphasize the duality of control, the results are presented as three-dimensional stereoscopic projections. Under physiological conditions, with 5.0 mM creatine, increases in Pi or decreases in phosphocreatine had little influence upon mitochondrial respiration. When phosphocreatine was held constant (15 mM), changes in [creatine] modestly stimulated respiratory rates, whereas Pi again showed little effect. With 1.0 mM Pi, respiration clearly became dependent upon changes in [creatine] and [phosphocreatine]. Initially, respiratory rates increased as a function of [creatine]. However, at [phosphocreatine] values below 10 mM, product "deinhibition" was observed, and respiratory rates rapidly increased to 80% State 3. With 2.0 mM Pi or higher, respiration could be regulated from State 4 to 100% State 3. Overall, the data show how increasing [creatine] and decreasing [phosphocreatine] influence the rates of oxidative phosphorylation when mediated by mitochondrial creatine kinase. Thus, these changes may become secondary cytoplasmic signals regulating heart oxygen consumption.     

Creatine kinase-catalyzed ATP-phosphocreatine exchange: comparison of 31P-NMR saturation transfer technique and radioisotope tracer methods

Unidirectional fluxes from ATP to phosphocreatine, catalyzed by the MM isoenzyme of creatine kinase, were measured by both the 31P-NMR saturation transfer technique and radioisotope tracer ([gamma-32P]ATP) method. It was found that at 30-37 degrees C and pH 7.4, over a wide range of [phosphocreatine]/[creatine] (from 0.2 to 5.0) ratios, both methods gave the same results, showing that magnetization transfer allows determination of real fluxes under 'physiological' conditions. However, at [PCr]/[Cr] ratios higher than 5 ([ADP]free less than 30 microM) or at lower temperatures (t less than 15 degrees C, [PCr]/[Cr] approximately 1), the fluxes assessed by saturation transfer were somewhat faster than those detected by the radioisotope tracer method. These data imply that under physiological conditions phosphoryl group transfer is actually the rate-determining step of the creatine kinase reaction. In contrast, at high [PCr]/[Cr] ratios or at lower temperatures, control may be shifted from phosphoryl group transfer or distributed among other steps of the reaction.     

A Novel Hypothesis About Mechanisms Affecting Conduction Velocity of Central Myelinated Fibers

The hypothesis that gap junctions are implicated in facilitating axonal conduction has not yet been experimentally demonstrated at the electrophysiological level. We found that block of gap junctions with oleammide slows down axonal conduction velocity in the hippocampal Schaffer collaterals, a central myelinated pathway. Moreover, we explored the possibility that support by the oligodendrocyte to the axon involves energy metabolism, a hypothesis that has been recently proposed by some of us. In agreement with this hypothesis, we found that the effect of oleammide was reversed by pretreatment with creatine, a compound that is known to increase the energy charge of the tissue. Moreover, conduction velocity was also slowed down by anoxia, a treatment that obviously decreases the energy charge of the tissue, and by ouabain, a compound that blocks plasma membrane Na/K-ATPase, the main user of ATP in the brain. We hypothesize that block of gap junctions slows down conduction velocity in central myelinated pathways because oligodendrocytes synthesize ATP and transfer it to the axon through gap junctions.     

Neuromodulatory effect of creatine on extracellular action potentials in rat hippocampus: role of NMDA receptors

The creatine (Cr) and phosphocreatine (PCr) system is essential for the buffering and transport of high-energy phosphates. Although achievements made over the last years have highlighted the important role of creatine in several neurological diseases, the adaptive processes elicited by this guanidino compound in hippocampus are poorly understood. In the present study, we showed that creatine (0.5-25mM) gradually increases the amplitude of first population spike (PS) and elicits secondary PS in stratum radiatum of the CA1 region, in hippocampal slices. Creatine also decreased the intensity of the stimulus to induce PS, when compared with hippocampal slices perfused with artificial cerebrospinal fluid (ACSF). The competitive NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (AP5; 100microM) attenuated creatine-induced increase of amplitude of PS and appearance of secondary PS, providing pharmacological evidence of the involvement of NMDA receptors in the electrophysiological effects of creatine. Accordingly, creatine (0.01-1mM) increased [3H]MK-801 binding to hippocampal membranes by 55%, further indicating that this compound modulates NMDA receptor function. These results implicate the NMDA receptor in amplitude and population spike increase elicited by creatine in hippocampus. Furthermore, these data suggest that this guanidino compound may also play a putative role as a neuromodulator in the brain, and that at least some of its effects may be mediated by an increase in glutamatergic function.     

Effect of calcium on brain metabolism in vitro

In attempts to distinguish between direct and indirect effects of Ca on brain cell metabolism, respiration, glycolysis, ATP, phosphocreatine, incorporation of [¹⁴C] leucine into protein, and accumulation of⁴⁵Ca was determined in brain slices. Incubation was carried out in normal salt-balanced medium, in high-potassiumor ouabain-containing medium under aerobic and anaerobic conditions. Calcium ions inhibited slightly glycolysis and respiration in normal medium and activated amino acid incorporation into proteins. Levels of ATP and phosphocreatine remained normal. These effects were interpreted as due to a stabilization of plasma membranes by Ca ions to prevent their spontaneous depolarization. Incubation of slices in high-potassium and ouabain media in aerobic conditions in the presence of Ca resulted in activation of respiration and glycolysis, decrease of ATP and phosphocreatine levels, and inhibition of amino acid incorporation into proteins. The disturbances in energy metabolism, caused by the respiration-linked Ca uptake in brain mitochondria and concomitant inhibition of oxidative phosphorylation, may lead to the inhibition of amino acid incorporation into proteins. An increase in Ca levels in the cytoplasm may only be expected in anaerobic conditions during the incubation in high-potassium and ouabain media. This is manifested by a direct inhibition of glycolysis by Ca ions and a drastic decrease of ATP and phosphocreatine in slices. The results suggest that stimulation of aerobic glycolysis and inhibition of anaerobic glycolysis by Ca may explain the unknown mechanism of the so-called reversed Pasteur effect of brain slices incubated in high-potassium media.     

ATP,creatine phosphate,and mechanical activity in rainbow trout myocardium under inhibition of glycolysis and cell respiration

Summary Twitch force and resting tension of electrically stimulated ventricular strips of rainbow trout were compared with tissue contents of phosphocreatine, creatine, and ATP. The phosphocreatine/total creatine ratio, which was used to assess the cytoplasmic phosphorylation potential, fell with the fraction of cell respiration that was inhibited by sodium cyanide and N₂. Concomitantly, twitch force decreased while resting tension tended to increase. This relation between phosphocreatine/total creatine and mechanical parameters became more prominent as glycolysis was increasingly inhibited by sodium iodoacetate. Furthermore, glycolytic inhibition was followed by a decrease in the ATP/phosphocreatine ratio. The latter effect was the same in 1% and 6% CO₂. Thus, it cannot be ascribed to an action of intracellular pH on the creatine kinase catalyzed reaction. Notably, resting tension as well as twitch force relative to ATP was augmented by glycolytic inhibition. The main conclusions are that in the presence of a decreased mitochondrial activity, glycolysis protects contractility not only by counteracting a lowering in high energy phosphates but also by supporting the ATP/phosphocreatine ratio. Apparently, the creatine kinase activity is insufficient to maintain ATP in equilibrium with phosphocreatine. In addition, glycolysis seems to elevate the level of free phosphate relative to ATP, so that twitch force development as well as rigor complex formation is counteracted.     

Hormone regulation of cardiac energy metabolism. I. Creatine transport across cell membranes of euthyroid and hyperthyroid rat heart

Hyperthyroid rat heart was studied with the purpose of identifying the mechanism for the significant decrease in total creatine (free creatine plus phosphocreatine) observed in this pathology and its consequences on heart function. Administration of L-thyroxine in doses of 50-100 micrograms/100 g of body weight during a week resulted in a reversible decrease of the total creatine by 40-50%. Simultaneously, remarkable changes in the creatine transport system across the cardiac cell membranes were observed: both the maximal rate of its active uptake and its passive movement along its concentration gradient were enhanced. In euthyroid hearts, the parameters of creatine uptake (Km approximately or equal to 0.05 mM, Vmax = 20 nmole/min/g dry weight) were similar to those for skeletal muscle and the passive movement of creatine was negligible. In hyperthyroid hearts the latter rate was enhanced to 0.4 mumole min/g dry weight, this showing reversible damages in the cell membrane structure induced by L-thyroxine. This conclusion is consistent with observed penetration of colloidal lanthanum into the cells of hyperthyroid hearts. Perfusion of hyperthyroid rat hearts with 50 mM creatine significantly restored creatine content in the cells, Hyperthyroid hearts with decreased creatine content were found to develop ischemic contracture more rapidly and in higher extent than the euthyroid hearts. Increased sensitivity to ischemic damage may be related to decreased efficiency of energy channeling via phosphocreatine pathway.     

Creatine kinase kinetics,ATP turnover,and cardiac performance in hearts depleted of creatine with the substrate analogue β-guanidinopropionic acid

Rats were fed a diet containing 1% of the creatine substrate analogue β-guanidinopropionic acid for 6–10 weeks. ³¹P-NMR investigation of isolated, glucose-perfused working hearts showed a 90% reduction in [phosphocreatine] from 22.2 to 2.5 μmol/g dry wt in guanidinopropionic acid-fed animals but no change in [P_i], [ATP], or intracellular pH. The unidirectional exchange flux in the creatine kinase reaction (direction phosphocreatine → ATP) was measured by saturation transfer NMR in hearts working against a perfusion pressure of 70 cm of water. This exchange was 10 μmol/g dry wt per s in control hearts and decreased 4-fold to 2.5–2.8 μmol/g dry wt per s in hearts from guanidinopropionic acid-fed animals. Oxygen consumption and cardiac performance were measured in parallel experiments at two perfusion pressures, 70 and 140 cm. No significant differences were observed in oxygen uptake or in any of the performance criteria between hearts from control and guanidinopropionic acid-fed rats at either workload. Assuming an ADP:O ratio of 3, the oxygen consumption measurements correspond to ATP turnover rates of 4.2–7.8 μmol/g dry per s. These rates are 1.5–3-times greater than the rate of the phosphocreatine → ATP exchange in hearts from guanidinopropionic acid-fed rats. These data suggest that phosphocreatine cannot be an obligate intermediate of energy transduction in the heart.     

Functional coupling of glycolysis and phosphocreatine utilization in anoxic fish muscle. An in vivo 31P NMR study

Three fish species with different strategies for anoxic survival (goldfish, tilapia, and common carp) were exposed to environmental anoxia (4, 3, and 1 h, respectively). The concentrations of high energy phosphate compounds and inorganic phosphate, besides the intracellular pH in the epaxial muscle were measured during anoxia and recovery by in vivo 31P NMR spectroscopy. The concentration of free ADP was calculated from the equilibrium constant of creatine kinase. During anoxia the patterns of phosphocreatine utilization and tissue acidification are remarkedly similar. Free ADP rises rapidly during the initial period of oxygen deficiency and reaches a plateau in goldfish and tilapia, while it keeps rising in the common carp. At elevated levels of free ADP, the creatine kinase reaction and anaerobic glycolysis are functionally coupled by H+ as a common intermediate. The coupling between both processes disappears upon reoxygenation, when mitochondrial respiration induces a rapid drop of [free ADP]. The removal of ADP shifts the creatine kinase equilibrium toward phosphocreatine synthesis despite the low pH.