In cerebral gray matter, ATP concentration is closely maintained despite rapid, large increases in turnover and low substrate reserves. As seen in vivo by (31)P nuclear magnetic resonance (NMR) spectroscopy, brain ATP is stable early in seizures, a state of high energy demand, and in mild hypoxia, a state of substrate deficiency. Like other tissues with high and variable ATP turnover, cerebral gray matter has high phosphocreatine (PCr) concentration and both cytosolic and mitochondrial creatine kinase (UbMi-CK) isoenzymes. To understand the physiology of brain creatine kinases, we used (31)P NMR to study PCr and ATP regulation during seizures and hypoxia in mice with targeted deletion of the UbMi-CK gene. The baseline CK reaction rate constant (k) was higher in mutants than wild-types. During seizures, PCr and ATP decreased in mutants but not in wild-types. The k-value for the CK catalyzed reaction rate increased in wild-types but not in the mutants. Hypoxic mutants and wild-types showed similar PCr losses and stable ATP. During recovery from hypoxia, brain PCr and ATP concentrations returned to baseline in wild-types but were 20% higher than baseline in the mutants. We propose that UbMi-CK couples ATP turnover to the CK catalyzed reaction rate and regulates ATP concentration when synthesis is increased.
The protective effects of creatine against glutamate cytotoxicity have been demonstrated in neuronal cells and animal models of neurodegenerative diseases. The mechanisms underlying creatine neuroprotection against glutamate-induced cell death are understood poorly. For the first time, we demonstrate a correlation between the protective effect of creatine and the modulation of Ras-mediated redox-dependent signaling pathways, which involve nuclear factor kappaB (NF-kappaB) and reactive oxygen species (ROS). In primary cerebrocortical cultures of mixed neurons and glia, creatine significantly reduced glutamate-induced cell death. The increase in cell survival was accompanied by increased generation of oxygen radicals and decreased levels of farnesylated Ras and IkappaB, an inhibitor of NF-kappaB. Non-farnesylated Ras and ROS-dependent activation of NF-kappaB have been shown to promote neuronal survival. Our data suggest that creatine may enhance survival signaling via activation of the Ras/NF-kappaB system. Possible mechanisms underlying the protective effect of creatine are discussed, including normalization of cellular GTP levels.
Systemic creatine (Cr) supplementation increases brain phosphocreatine (PCr) and prevents hypoxic seizures in 15-day-old rabbits . Between 5 and 30 days of age during normal development, rabbit gray matter mitochondrial creatine kinase (Mi-CK) increases 400% while cytosolic CK (BB-CK) increases 60%. In white matter, both isoenzymes show smaller, similar increases (40%) during this period. The Cr transporter protein decreases 60% between 5 and 15 days in both regions. In vivo CK rate constants measured by 31P nuclear magnetic resonance increase 30% between 10 and 20 days, and then fall 50% between 20 and 30 days in predominantly gray matter slices. Similar maturational changes are seen in predominantly white matter slices. Injecting Cr at 15 days does not significantly change BB-CK or Mi-CK isoenzymes or the in vivo CK reaction rate constants. Thus, the largest change in the CK system associated with suppression of hypoxic seizures in Cr-treated rabbits is increased PCr in gray and white matter.
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