Creatine ethyl ester: A new substrate for creatine kinase

The creatine kinase/phosphocreatine system plays a key role in cell energy buffering and transport, particularly in cells with high or fluctuating energy requirements, like neurons, i.e. it participates in the energetic metabolism of the brain. Creatine depletion causes several nervous system diseases, alleviated by phosphagen supplementation. Often, the supplementation contains both creatine and creatine ethyl ester, known to improve the effect of creatine through an unknown mechanism. In this work we showed that purified creatine kinase is able to phosphorilate the creatine ethyl ester. The K _m and V _max values, as well as temperature and pH optima were determined. Conversion of the creatine ethyl ester into its phosphorylated derivative, sheds light on the role of the creatine ethyl ester as an energy source in supplementation for selected individuals.     

Interaction of creatine kinase and adenylate kinase systems in muscle cells

Elsewhere in this book the important role of creatine kinase and its metabolites in high energy phosphate metabolism and transport in muscle cells has been reviewed. The emphasis of this review article is mainly on the compartmentalized catalytic activity of adenylate kinase in relation to creatine kinase isoenzymes, and other enzymes of energy production and utilization processes in muscle cells. At present the role of adenylate kinase is considered simply to equilibrate the stores of adenine nucleotides. Recent studies by us and others, however, suggest an entirely new view of the metabolic importance of adenylate kinase in muscle function. This view offers a closer interaction between adenylate kinase and creatine kinase, in the process of energy production (at mitochondrial and glycolytic sites), and energy utilization (at myofibrillar sites and perhaps other sites such as sarcoplasmic reticular, sarcolemmal membrane, etc.), thus being an integral part of the high energy phosphate transport system.This review article opens up the opportunity to further examine the metabolism of adenine nucleotides and their fluxes through the adenylate kinase system in intact muscle cells. Using an intact system, having a preserved integrity of their compartmentalized enzymes and substrates, is essential in clarifying the exact role of adenylate kinase in high energy phosphate metabolism in muscle cells.     

The polysome as a terminal for the creatine phosphate energy shuttle

The role of the creatine phosphate shuttle in the energetics of muscle protein synthesis in isolated polysomes, from rat hindlimb muscle, was studied. Triton X-100-treated polysomes, following their centrifugation through a 1 M sucrose gradient, contained 38 mU/mg RNA of bound creatine kinase. In the presence of pH 5 enzyme (obtained from rat liver), 0.5 mM ATP, and 1 microM GTP, amino acid (leucine) incorporation by polysomes in the presence of 8 mM creatine phosphate was twice that in the presence of an exogenous ATP regenerating system of 10 mM phospho(enol)pyruvate and 10 U/ml pyruvate kinase. Since added creatine kinase had no effect on incorporation supported by creatine phosphate it is clear that endogenous creatine kinase allows sufficient regeneration of ATP. These data also suggest that nucleoside diphosphokinase must have been associated with the polysome for phosphate was transferred to GTP from [33P]creatine phosphate, and the specific activities of ATP and GTP increased at equal rates, reaching the specific activity of creatine phosphate at 8 min. We conclude that skeletal muscle polysomes have bound creatine kinase activity and they act as terminals for the creatine phosphate energy shuttle. Creatine phosphate regenerates GTP, probably through an intermediate reaction catalyzed by nucleoside diphosphokinase. This provided an added support for the hypothesis of compartmentation of enzymes and substrates and that the transport form of energy between the mitochondria and energy utilizing sites in muscle is creatine phosphate rather than ATP, which extends the general role of the creatine phosphate energy shuttle.     

A new method to synthesize creatine derivatives

Creatine is an amino acid that has a pivotal role in energy metabolism of cells. Creatine acts as an “ATP shuttle”, carrying ATP to the sites where it is utilized, through its reversible phosphorylation by creatine kinase. Moreover, the creatine-phosphocreatine system delays ATP depletion during anoxia or ischemia, thus exerting a neuroprotective role during those pathological conditions. Thus, its administration has been advocated as a treatment or prevention of several conditions involving the central nervous system. However, creatine crosses poorly the blood–brain barrier and the cell plasma membrane, thus its administration has but a limited effect. The use of more lipophilic creatine derivatives has thus been suggested. However, such a synthesis is complicated by the intrinsic characteristics of the creatine molecule that hardly reacts with other molecules and easily cyclizes to creatinine. We obtained amide derivatives from creatine starting from a new protected creatine molecule synthesized by us, the so-called (Boc)₂-creatine. We used a temporary protection only on the creatine guanidine group while allowing a good reactivity on the carboxylic group. This temporary protection ensured efficient creatine dissolution in organic solvents and offered simultaneous protection of creatine toward intramolecular cyclization to creatinine. In this manner, it was possible to selectively conjugate molecules on the carboxylic group. The creatine guanidine group was easily released from the protection at the end of the reaction, thus obtaining the desired creatine derivative.     

In vivo and in vitro assessment of brain bioenergetics in aging rats

Brain energy disorders can be present in aged men and animals. To this respect, the mitochondrial and free radical theory of aging postulates that age‐associated brain energy disorders are caused by an imbalance between pro‐ and anti‐oxidants that can result in oxidative stress. Our study was designed to investigate brain energy metabolism and the activity of endogenous antioxidants during their lifespan in male Wistar rats. In vivo brain bioenergetics were measured using ³¹P nuclear magnetic resonance (NMR) spectroscopy and in vitro by polarographic analysis of mitochondrial oxidative phosphorylation. When compared to the young controls, a significant decrease of age‐dependent mitochondrial respiration and adenosine‐3‐phosphate (ATP) production measured in vitro correlated with significant reduction of forward creatine kinase reaction (kfor) and with an increase in phosphocreatine (PCr)/ATP, PCr/Pi and PME/ATP ratio measured in vivo. The levels of enzymatic antioxidants catalase, GPx and GST significantly decreased in the brain tissue as well as in the peripheral blood of aged rats. We suppose that mitochondrial dysfunction and oxidative inactivation of endogenous enzymes may participate in age‐related disorders of brain energy metabolism.     

Comparison of the two different phosphagen systems in the lugworm Arenicola marina

The different phosphagen systems in the lugworm Arenicola marina, the phosphotaurocyamine/taurocyamine kinase system of the body wall and the phosphocreatine/creatine kinase system of the spermatozoa, have been investigated to answer the question whether the change reflects different functional modes of these phosphagen systems. Enzyme analyses have shown that in contrast to the body wall taurocyamine kinase, creatine kinase of spermatozoa exists in at least two different forms which are compartmented in the mitochondria (creatine kinase I) and in the flagellum (creatine kinase II). Creatine kinase I is strongly attached to cell structures which require detergents and high phosphate concentrations for solubilization. The affinities of taurocyamine kinase and creatine kinase for all substrates are very similar except the extremely high K _m for creatine of both creatine kinase I and II. The level of creatine in spermatozoa is fivefold higher than taurocyamine in the body wall at similar phosphorylation potential (ATP/ADO_free) and ATP-buffer capacity (phosphagen/ATP), reflecting the higher equilibrium constants of the creatine kinase reaction compared to that of the taurocyamine kinase reaction (Ellington 1989). The high creatine concentration gives the phosphocreatine/creatine kinase system an advantage over the phosphotaurocyamine/taurocyamine kinase system for transport of energyrich phosphate at high phosphorylation potential by increasing the radial diffusion flux. The maximum diffusive flux of free ADP in spermatozoa is three orders of magnitude below the respiratory ATP production while the creatine flux would allow an unlimited energy transport over the long diffusion distance. In lugworm body wall, however, the low ATP turnover and the low diffusion distances between mitochondria and myosin-ATPases do not require a phosphagen shuttle.Abbreviations ADP _free cytoplasmic adenosine diphosphate - Ap ₅ A P₁, P₅-di(adenosine-5-) pentaphosphate - AK arginine kinase - CK creatine kinase (EC 2.7.3.2) - DTT dithiothreitol - GAPDH glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) - HOADH 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) - IEP isoelectric point - MIM mitochondria isolating medium - P _i-free cytoplasmic inorganic phosphate - (P)Arg (phospho)arginine - (P)Cr (phospho)creatine - (P)Tc (phospho)taurocyamine - SEM scanning electron microscopy - TK taurocyamine kinase - TEM transmission electron microscopy     

The effect of inorganic phosphate on creatine kinase in respiring rat heart mitochondria

The ability of creatine to stimulate the respiration of rat heart mitochondria in vitro is reversibly affected by the concentration of inorganic phosphate. The rate of oxygen consumption due to post-ADP state-4 respiration in the presence of 20 mm creatine is reduced significantly when the potassium phosphate concentration is raised from 5 to 20 mm. State-3 respiration is reduced only by potassium phosphate concentrations higher than 20 mm. The rate of synthesis of creatine phosphate is also affected by phosphate concentration, and the apparent K_m of the coupled reactions for ADP is significantly higher at 25 mm phosphate as compared to that at 5 mm phosphate. These observations are consistent with the hypothesis that inorganic phosphate acts as an effector molecule, regulating creatine phosphate synthesis by favoring the dissociation of mitochondrial creatine kinase from the mitochondrial membrane. Such regulation may be important in the case of cells undergoing partial or severe ischemia, where changes in phosphate concentration within this range have been reported.     

Metabolic alterations associated with increased energy demand in fish white muscle

Summary Time course measurements of glycogen, lactate, creatine phosphate, the adenylates and ammonia contents were made during the transition from rest to various levels of activity in fish (Macrozoarces americanus) white muscle. The muscle was perturbed by direct electrical stimulation resulting in sustained tetanus, 60 contractions/min or 20 contractions/min. Increased ATP demand was invariably associated with decreases in creatine phosphate followed by increases in lactate levels. The contribution of creatine phosphate to anaerobic energy production was equivalent to that of anaerobic glycolysis. In addition, decreases in creatine phosphate content may play an important role in the facilitation of glycolytic flux presumably by relief of inhibition of phosphofructokinase. Under some conditions the work transition was associated with an initial transient increase in ATP content which could not be accounted for by decreases in ADP and AMP levels. Furthermore, ammonia content was noted to oscillate during the work period, a feature which is fundamentally different from that which occurs in mammalian muscle.     

Isolation and characterization of mutants of the facultative methylotroph Arthrobacter P1 blocked in one-carbon metabolism

Among methylamine and/or ethylamine minus mutants of Arthrobacter P1 four different classes were identified, which were blocked either in the methylamine transport system, amine oxidase, hexulose phosphate synthase or acetaldehyde dehydrogenase. The results indicated that a common primary amine oxidase is involved in the metabolism of methylamine and ethylamine. Growth on ethylamine, however, was not dependent on the presence of the methylamine transport system. In mutants lacking amine oxidase, methylamine was unable to induce the synthesis of hexulose phosphate synthase, thus confirming the view that the actual inducer for the latter enzyme is not methylamine, but its oxidation product formaldehyde. Contrary to expectation, when the formaldehyde fixing enzyme hexulose phosphate synthase was deleted (mutant Art 11), accumulation of formaldehyde during growth on choline or on glucose plus methylamine as a nitrogen source did not occur. Evidence was obtained to indicate that under these conditions formaldehyde may be oxidized to carbon dioxide via formate, a sequence in which peroxidative reactions mediated by catalase are involved. In addition, a specific NAD-dependent formaldehyde dehydrogenase was detected in choline-grown cells of wild type Arthrobacter P1 and strain Art 11. This enzyme, however, does not play a role in methylamine or formaldehyde metabolism, apparently because these compounds do not induce its synthesis.Abbreviations RuMP ribulose monophosphate - HPS hexulose phosphate synthase - HPI hexulose phosphate isomerase     

Construction of an efficient Escherichia coli cell‐free system for in vitro expression of several kinds of proteins

Cell‐free protein synthesis systems have offered several advantages over traditional cell‐based expression methods. In this study, the effects of extract preparation and an energy‐regenerating system on protein synthesis were investigated in an Escherichia coli cell‐free system. The results indicated that the expression level of enhanced green fluorescent protein (eGFP) with the S12 extract was higher than that with the S30 extract. Among four adenosine triphosphate‐regenerating sources, the cAMP/CP/CK system (including cAMP, creatine phosphate, and creatine kinase) proved to be the most efficient one to support high‐level expression of eGFP. Further studies showed that this established cell‐free system could be successfully used to produce one model protein (eGFP), two human proteins (AK2 and coenzyme synthase) and two membrane proteins (subunit b of F₁F₀ adenosine triphosphate synthase and aquaporin Z). This outcome will be helpful to develop the highly efficient cell‐free technology for the production of various proteins with different bio‐origins.     

Creatine kinase and mitochondrial respiration in hearts of trout,cod and freshwater turtle

The importance of the creatine kinase system in the cardiac muscle of ectothermic vertebrates is unclear. Mammalian cardiac muscle seems to be structurally organized in a manner that compartmentalizes the intracellular environment as evidenced by the substantially higher mitochondrial apparent K_m for ADP in skinned fibres compared to isolated mitochondria. A mitochondrial fraction of creatine kinase is functionally coupled to the mitochondrial respiration, and the transport of phosphocreatine and creatine as energy equivalents of ATP and ADP, respectively, increases the mitochondrial apparent ADP affinity, i.e. lowers the K_m. This function of creatine kinase seems to be absent in hearts of frog species. To find out whether this applies to hearts of ectothermic vertebrate species in general, we investigated the effect of creatine on the mitochondrial respiration of saponin-skinned fibres from the ventricle of rainbow trout, Atlantic cod and freshwater turtle. For all three species, the apparent K_m for ADP appeared to be substantially higher than for isolated mitochondria. Creatine lowered this K_m in trout and turtle, thus indicating a functional coupling between mitochondrial creatine kinase and respiration. However, creatine had no effect on K_m in cod ventricle. In conclusion, the creatine kinase-system in trout and turtle hearts seems to fulfil the same functions as in the mammalian heart, i.e. facilitating energy transport and communication between cellular compartments. In cod heart, however, this does not seem to be the case.Abbreviations ACR acceptor control ratio - CK creatine kinase - PCr creatine phosphate - V_ADP ADP-stimulated respiration rate - V_max maximal respiration rate - V₀ respiration rate in the absence of ADPCommunicated by: G. Heidmaier     

Intimate coupling of creatine phosphokinase and myofibrillar adenosinetriphosphatase

ATPase and creatine phosphokinase (CPK) activities of isolated cardiac myofibrils were determined with ³²P γ-labeled ATP alone and with the addition of phosphorylcreatine (PC). With ATP and PC as substrates the label in the inorganic phosphate formed is greatly diluted indicating that the ATP formed by PC through CPK can reach the ATPase active site more readily than labeled ATP from the medium. The tight coupling of the ATPase and CPK activities further strengthens our view that PC serves an important role as high energy carrier between the energy producing sites (mitochondria) and the energy utilizing sites (myofibrils).     

Creatine ethyl ester: a new substrate for creatine kinase

The creatine kinase/phosphocreatine system plays a key role in cell energy buffering and transport, particularly in cells with high or fluctuating energy requirements, like neurons, i.e. it participates in the energetic metabolism of the brain. Creatine depletion causes several nervous system diseases, alleviated by phosphagen supplementation. Often, the supplementation contains both creatine and creatine ethyl ester, known to improve the effect of creatine through an unknown mechanism. In this work we showed that purified creatine kinase is able to phosphorilate the creatine ethyl ester. The K(m) and V(max) values, as well as temperature and pH optima were determined. Conversion of the creatine ethyl ester into its phosphorylated derivative, sheds light on the role of the creatine ethyl ester as an energy source in supplementation for selected individuals.     

Phosphocreatine production coupled to the glycolytic reactions in the cytosol of cardiac cells

Phosphocreatine production catalyzed by a cytosolic fraction from cardiac muscle containing all glycolytic enzymes and creatine kinase in a soluble form has been studied in the presence of creatine, adenine nucleotides and different glycolytic intermediates as substrates. Glycolytic depletion of glucose, fructose 1,6-bis(phosphate) and phosphoenolpyruvate to lactate was coupled to efficient phosphocreatine production. The molar ratio of phosphocreatine to lactate produced was close to 2.0 when fructose 1,6-bis(phosphate) was used as substrate and 1.0 with phosphoenolpyruvate. In these processes the creatine kinase reaction was not the rate-limiting step: the mass action ratio of the creatine kinase reaction was very close to its equilibrium value and the maximal rate of the forward creatine kinase reaction exceeded that of glycolytic flux by about 6-fold when fructose 1,6-bis(phosphate) was used as a substrate. Therefore, the creatine kinase raction was continuously in the state of quasiequilibrium and the efficient synthesis of phosphocreatine observed is a result of constant removal of ADP by the glycolytic system at an almost unchanged level of ATP ([ATP] ? [ADP]), this leading to a continuous shift of the creatine kinase equilibrium position.When phosphocreatine was added initially at concentrations of 5–15 mM the rate of the coupled creatine kinase and glycolytic reactions was very significantly inhibited due to a sharp decrease in the steady-state concentration of ADP. Therefore, under conditions of effective phosphocreatine production in heart mitochondria, which maintain a high phosphocreatine: creatine ratio in the myoplasm in vivo, the glycolytic flux may be suppressed due to limited availability of ADP restricted by the creatine kinase system. The possible physiological role of the control of the glycolytic flux by the creatine kinase system is discussed.     

A P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

Influence of mitochondrial creatine kinase on the mitochondrial/extramitochondrial distribution of high energy phosphates in muscle tissue: evidence for a leak in the creatine shuttle

The influence of mitochondrial creatine kinase on subcellular high energy systems has been investigated using isolated rat heart mitochondria, mitoplasts and intact heart and skeletal muscle tissue.In isolated mitochondria, the creatine kinase is functionally coupled to oxidative phosphorylation at active respiratory chain, so that it catalyses the formation of creatine phosphate against its thermodynamic equilibrium. Therefore the mass action ratio is shifted from the equilibrium ratio to lower values. At inhibited respiration, it is close to the equilibrium value, irrespective of the mechanism of the inhibition. The same results were obtained for mitoplasts under conditions where the mitochondrial creatine kinase is still associated with the inner membrane.In intact tissue increasing amounts of creatine phosphate are found in the mitochondrial compartment when respiration and/or muscle work are increased. It is suggested that at high rates of oxidative phosphorylation creatine phosphate is accumulated in the intermembrane space due to the high activity of mitochondrial creatine kinase and the restricted permeability of reactants into the extramitochondrial space. A certain amount of this creatine phosphate leaks into the mitochondrial matrix.This leak is confirmed in isolated rat heart mitochondria where creatine phosphate is taken up when it is generated by the mitochondrial creatine kinase reaction. At inhibited creatine kinase, external creatine phosphate is not taken up. Likewise, mitoplasts only take up creatine phosphate when creatine kinase is still associated with the inner membrane. Both findings indicate that uptake is dependent on the functional active creatine kinase coupled to oxidative phosphorylation.Creatine phosphate uptake into mitochondria is inhibited with carboxyatractyloside. This suggests a possible role of the mitochondrial adenine nucleotide translocase in creatine phosphate uptake.Taken together, our findings are in agreement with the proposal that creatine kinase operates in the intermembrane space as a functional unit with the adenine nucleotide translocase in the inner membrane for optimal transfer of energy from the electron transport chain to extramitochondrial ATP-consuming reactions.     

Transient alterations of creatine, creatine phosphate, N-acetylaspartate and high-energy phosphates after mild traumatic brain injury in the rat

In this study, the concentrations of creatine (Cr), creatine phosphate (CrP), N-acetylaspartate (NAA), ATP, ADP and phosphatidylcholine (PC) were measured at different time intervals after mild traumatic brain injury (mTBI) in whole brain homogenates of rats. Anaesthetized animals underwent to the closed-head impact acceleration “weight-drop” model (450 g delivered from 1 m height = mild traumatic brain injury) and were killed at 2, 6, 24, 48 and 120 h after the insult (n = 6 for each time point). Sham-operated rats (n = 6) were used as controls. Compounds of interest were synchronously measured by HPLC in organic solvent deproteinized whole brain homogenates. A reversible decrease of all metabolites but PC was observed, with minimal values recorded at 24 h post-injury (minimum of CrP = 48 h after impact). In particular, Cr and NAA showed a decrease of 44.5 and 29.5%, respectively, at this time point. When measuring NAA in relation to other metabolites, as it is commonly carried out in “in vivo” ¹H-magnetic resonance spectroscopy (¹H-MRS), an increase in the NAA/Cr ratio and a decrease in the NAA/PC ratio was observed. Besides confirming a transient alteration of NAA homeostasis and ATP imbalance, our results clearly show significant changes in the cerebral concentration of Cr and CrP after mTBI. This suggests a careful use of the NAA/Cr ratio to measure NAA by ¹H-MRS in conditions of altered cerebral energy metabolism. Viceversa, the NAA/PC ratio appears to be a better indicator of actual NAA levels during energy metabolism impairment. Furthermore, our data suggest that, under pathological conditions affecting the brain energetic, the Cr–CrP system is not a suitable tool to buffer possible ATP depletion in the brain, thus supporting the growing indications for alternative roles of cerebral Cr.     

Hair bundles are specialized for ATP delivery via creatine kinase

When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those proteins participating in energy metabolism, we used shotgun mass spectrometry methods to identify proteins of purified chicken vestibular bundles. In addition to cytoskeletal proteins, proteins involved in Ca(2+) regulation, and stress-response proteins, many of the most abundant bundle proteins that were identified by mass spectrometry were involved in ATP synthesis. After beta-actin, the cytosolic brain isoform of creatine kinase was the next most abundant bundle protein; at approximately 0.5 mM, creatine kinase is capable of maintaining high ATP levels despite 1 mM/s ATP consumption by the plasma-membrane Ca(2+)-ATPase. Consistent with this critical role in hair bundle function, the creatine kinase circuit is essential for high-sensitivity hearing as demonstrated by hearing loss in creatine kinase knockout mice.     

Determiantion of creatine phosphate levels in brain tissue by isocratic reverse-phase,ion-paired high-performance liquid chromatography

The utilization of isocratic, reverse-phase, ion-paired high-performance liquid chromatography for analysis of creatine phosphate allows for rapid quantification of multiple samples. Cryogenic sample handling and the addition of ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid as a Ca²⁺ sequestering agent during perchloric acid extraction enhance maximal recovery of creatine phosphate from brain samples. Peak identification is supported by a complete enzymatic shift with a phosphocreatine kinase, hexokinase, and glucose-6-phosphate dehydrogenase system.     

Cell-Type-Specific Spatiotemporal Expression of Creatine Biosynthetic Enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase in Developing Mouse Brain

Creatine is synthesized by S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT), and the creatine/phosphocreatine shuttle system mediated by creatine kinase (CK) is essential for storage and regeneration of high-energy phosphates in cells. Although the importance of this system in brain development is evidenced by the hereditary nature of creatine deficiency syndrome, the spatiotemporal cellular expression patterns of GAMT in developing brain remain unknown. Here we show that two waves of high GAMT expression occur in developing mouse brain. The first involves high expression in mitotic cells in the ventricular zone of the brain wall and the external granular layer of the cerebellum at the embryonic and neonatal stages. The second was initiated by striking up-regulation of GAMT in oligodendrocytes during the second and third postnatal weeks (i.e., the active myelination stage), which continued to adulthood. Distinct temporal patterns were also evident in other cell types. GAMT was highly expressed in perivascular pericytes and smooth muscle cells after birth, but not in adults. In neurons, GAMT levels were low to moderate in neuroblasts residing in the ventricular zone, increased during the second postnatal week when active dendritogenesis and synaptogenesis occur, and decreased to very low levels thereafter. Moderate levels were observed in astrocytes throughout development. The highly regulated, cell type-dependent expression of GAMT suggests that local creatine biosynthesis plays critical roles in certain phases of neural development. In accordance with this idea, we observed increased CK expression in differentiating neurons; this would increase creatine/phosphocreatine shuttle system activity, which might reflect increased energy demand.