Creatine and creatinine transport in old and young human red blood cells

The time course of creatine influx or efflux as measured in populations of red cells or red cell ghosts with normal age distribution does not follow simple two-compartment kinetics. This suggests that the contributions of individual cells to transport as measured in the populations as a whole are not uniform. In agreement with this inference, fractionation of red cell populations with respect to cell age shows that transport in young cells is considerably faster than in old cells.The dependence of creatine transport on creatine concentration in the medium follows an equation that can be interpreted to represent a super-imposition of a saturable component (apparent K_m = 0.02 mM) and another component that cannot be saturated up to a creatine concentration of 5.0 mM. In contrast to the non-saturable component, the saturable component depends on the energy metabolism of the cell and can be inhibited by β-guanidinopropionic acid and the proteolytic enzyme pronase. This latter finding suggests that the saturable component represents active transport that is mediated by a transport protein. The non-saturable component is little, if at all, dependent on cell age while the saturable component is higher in young cells than in old cells. Phloretin inhibits both components of creatine flux, but the maximal inhibition that can be achieved at high concentration is only 70–80%.Under the experimental conditions used for the study of creatine transport, creatinine equilibration between cells and medium follows the kinetics expected for a steady-state two-compartment system. Creatinine flux is proportional to creatine concentration over the concentration range studied (up to 5 mM). It cannot be inhibited by β-guanidinopropionic acid or pronase.     

Changes in red cell density and related indices in response to distance running

The red cell population in peripheral venous blood was characterised in 7 young males before and up to 16 days after a 21.1 km road race. There was a 1.9 +/- 2.4% (mean +/- SD) reduction in plasma volume immediately post race (p less than 0.05), an increase in serum osmolality from 277 +/- 4 mOsm.kg-1 to 291 +/- 14 mOsm.kg-1 (p less than 0.05) and a reduction in red cell water (64.4 +/- 0.3% to 63.4 +/- 0.4%, p less than 0.001). The latter was consistent with alterations in the manually derived MCV and MCHC values although the same Coulter derived values were unaltered. A concomitant increase in median red cell density in whole blood (1.1045 +/- 0.0009 g.ml-1 pre race to 1.1057 +/- 0.012 g.ml-1 immediate post race, p less than 0.05) was recorded by centrifugation through phthalate esters of different density. The changes in creatine content of the red cells suggested that during the race younger cells were released into the circulation but that 24 h to 72 h after the race the mean red cell age had increased. Similarly, fractionation of the red cells on discontinuous Percoll density gradients indicated that the cell population was significantly denser in all post race samples up to 72 h but had normalized by a 16 day sample; the osmotic fragility was similarly affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of creatine, creatinine, creatine-d3 and creatinine-d3 in urine, plasma, and red blood cells by HPLC and GC-MS to follow the fate of ingested creatine-d3

Creatine, which is increasingly being used as an oral supplement, is naturally present in the body. Studies on the fate of a particular dose of creatine require that the creatine be labeled, and for studies in humans the use of a stable isotopic label is desirable. The concentrations of total creatine and total creatinine were determined using HPLC. Creatine and creatinine were then separated using cation exchange chromatography and each fraction was derivatized with trifluoroacetic anhydride and the ratio of the deuterated:undeuterated species determined using GC-MS. Ratios of creatine:creatine-d(3), and creatinine:creatinine-d(3), and the concentrations of each of these species, were able to be determined in urine, plasma and red blood cells. Thus, the uptake of labeled creatine into plasma and red blood cells and its excretion in urine could be followed for a subject who ingested creatine-d(3). Creatine-d(3) was found in the plasma and red blood cells 10 min after ingestion, while creatine-d(3) and creatinine-d(3) were found in the urine collected after the first hour.     

Creatine and density of red blood cells in perinatal hypoxia

Signs of stimulated erythropoiesis, such as increased creatine and decreased density of red cells are good indicators of hypoxemia in adults and older children. The sensitivity of both tests in perinatal hypoxia was found to be reduced. The causes for this reduction were investigated.     

Erythrocyte aging in neurodegenerative disorders.

In the present paper, we have reviewed the principal studies on red cell membrane abnormalities associated with neurodegenerative disorders. In the literature, two lines of investigation may be recognized: one based on the hypothesis of the presence of an oxidative environment responsible for red cell oxidative damage in Alzheimer's disease (AD), Alzheimer's dementia type (DAT) and Parkinson' disease (PD); the other one based on the identification of structural and/or functional abnormalities in red cell membrane band 3 and/or in red cell membrane lipid composition in "neuroacanthocytosis". In AD, DAT and PD patients, an increased red cell membrane lipid peroxidation suggests an increase red cell oxidative damages and precocious red cell aging. In "neuroacanthocytosis", grouping chorea-acanthocytosis, Mcleod syndrome and abetalipoproteinemia, the red cells are characterized by thorn or spur-like protrusions, known as "acanthocytes". The presence of circulating acanthocytes, characterized by abnormalities in red cell band 3 structure and/or function, is associated with increase levels of anti-band 3 antibodies which are physiologically produced against aged red cells and are known to mediate red cell removal from the peripheral circulation by macrophages. We have reviewed the mechanism(s) of the loss of red cell membrane stability and of the precocious red cell aging in neurodegenerative disorders.     

Regulation of creatine phosphokinase expression during differentiation of BC3H1 cells

The intracellular mechanisms involved in the regulation of creatine phosphokinase expression in the BC3H1 muscle-like cell line have been examined under conditions of enzyme induction and repression. In the presence of low serum concentrations, BC3H1 cells cease to grow and synthesize high levels of creatine phosphokinase. When differentiated BC3H1 cultures are exposed to media containing high serum concentrations, cell division is reinitiated and further induction of creatine phosphokinase is inhibited. Accumulation of creatine phosphokinase-mRNA appears to be intimately coupled to the state of growth of BC3H1 cells. Log phase cells do not contain detectable levels of translatable creatine phosphokinase-mRNA; however, following cessation of growth, creatine phosphokinase-mRNA accumulates in approximate proportion to the increase in creatine phosphokinase activity. Reinitiation of cell division in quiescent differentiated cultures results in the arrest of further accumulation of creatine phosphokinase-mRNA but does not inhibit the translation of pre-existing creatine phosphokinase-mRNA. Under conditions of enzyme repression, however, the newly synthesized creatine phosphokinase appears to be enzymatically inactive. These results indicate that the expression of the muscle phenotype in BC3H1 cells is regulated by components present in serum and that myogenic differentiation is at least partially reversible following re-entry of quiescent cells into the cell cycle.     

Changes in transferrin during the red cell replacement in amphibia

Transferrin, a plasma glycoprotein, carries iron from storage sites to immature erythroid cells for hemoglobin synthesis. The replacement of larval red cells by adult red cells, which occurs during metamorphosis in bullfrogs, requires extensive formation of hemoglobin and new red cells. Large changes in red cell iron storage also occur during the red cell replacement. Both the concentration and the level of iron saturation of plasma transferrin were measured during metamorphosis to determine if there were changes in plasma transferrin which coincided with the changes in red cell iron storage and ferritin content. Plasma transferrin concentrations increased from 0.96 to 2.6 mg/ml during the period when red cell storage iron and ferritin decreased. Plasma iron concentrations also increased when the transferrin concentration increased, suggesting that the additional transferrin may be involved in moving iron from the larval red cell stores. At the end of metamorphosis, the plasma iron concentration decreased to premetamorphic levels but the transferrin concentration remained high, resulting in a decrease in saturation to 18% compared to 45% in the larvae. In addition to differences in iron saturation, adult transferrin had different electrophoretic properties from larval transferrin. The results support the hypotheses that during early ontogeny plasma transferrin and red cell iron storage are coordinated to provide iron for the formation of the first generation of adult red cells and that transferrin may participate in the control of red cell ferritin synthesis.     

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.

     

Metabolism of Glycine in Primary Astroglial Cells: Synthesis of Creatine, Serine, and Glutathione

Abstract: The metabolism of [2-¹³C]glycine in astrogliarich primary cultures obtained from brains of neonatal Wistar rats was investigated using ¹³C NMR spectroscopy. After a 24-h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2-¹³C]glycine, cell extracts and incubation media were analyzed for ¹³C-labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2-¹³C]glycine, the amount of de novo synthesized [2-¹³C]creatine was two-fold increased, and in addition, ¹³C-labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2-¹³C]glycine was utilized for the synthesis of glutathione in astroglial cells. ¹³C-labeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2-¹³C]serine, [3-¹³C]serine, and [2,3-¹³C]serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons.     

Investigations on the function of creatine kinase in Ehrlich ascites tumor cells

1. Growth and viability of in vitro cultured Ehrlich ascites tumor cells are not significantly impaired by exogenous creatine up to 40mM. Retardation of cell growth by higher concentrations depends on cell density. 2. Ehrlich cells grown in the presence of high concentrations of creatine accumulate creatine phosphate to high levels (up to 23 nmol/10(6) cells in the presence of 40mM creatine). 3. A nearly complete interruption of glycolytic ATP production or inhibition of the oxidative ATP synthesis reduces the maximal creatine to about 40-50% of controls. 4. Studies on the intracellular distribution of creatine kinase have shown, that the enzyme is only associated with the mitochondrial fraction. Titration of isolated mitochondria with digitonin revealed that the activity is located in the inter-membrane space and partly bound to the outer site of the inner membrane. 5. By growth of Ehrlich cells in creatine-free medium it is possible to obtain "creatine phosphate-depleted" cells (creatine phosphate less than 10% of controls). The growth of creatine phosphate-depleted cells as compared to controls is significantly reduced under energetic stress situations. The protein synthesis of these cells after an energetic stress (lack of glucose and oxygen) is significantly reduced as compared to creatine phosphate containing cells. 6. It is concluded that in these cells creatine kinase/creatine phosphate is a thermodynamic buffer system and not part of an energy shuttle as is postulated for muscle cells.     

Crosslinking of membrane proteins in red blood cells from vitamin E-deficient lead-poisoned rats

Red blood cells from vitamin E-deficient rats lose their filterability after incubation in vitro and concurrent lead poisoning of the rats accelerates this decline. This decreased red cell filterability is associated with an increased crosslinking of red cell membrane proteins. Previous reports by others suggested an association between red cell glutathione levels and filterability, but we found no such association in red cells treated with N-ethylmaleimide. Increased aggregation of red cell membrane proteins may play a role in the discocyte/ spherocyte shape change that accounts for the impaired filterability of red blood cells from vitamin E-deficient lead-poisoned rats.     

Changes in membrane properties of rat red blood cells induced by cadmium accumulating in the membrane fraction

When rat red blood cells were incubated in a cadmium (Cd)-free medium following 1-h pretreatment with 0.5 mM CdCl2, incorporated Cd was retained in the cell during 14-h incubation and progressively accumulated in the membrane fraction, especially in the cytoskeleton fraction. In parallel to this accumulation, red cell filterability decreased and echinocytic cells increased, although intracellular ATP was maintained at the control level. The echinocytic shape was maintained in ghosts and cytoskeletons prepared from the Cd-loaded cells. In addition, the association of bands 2.1, 3, 4.2, and 4.5 with cytoskeletons increased and dissociation of cytoskeletal networks at low ionic strength decreased as the incubation time increased. Pretreatment of red blood cells with Cd also induced a release of small vesicles. These vesicles contained hemoglobin but were depleted of spectrin and actin, showing a phospholipid composition similar to that of red cell ghosts. These results suggest that the organization of cell membranes, especially cytoskeletal networks, is altered by Cd accumulation in the cytoskeleton fraction, which results in acceleration of age-related changes of red blood cells such as shape change and decreased filterability.     

Isozymes of creatine kinase in mammalian cell culures

Previous studies on the energy metabolism of rat myocardial cells in culture supported the hypothesis that the creatine-phosphocreatine–creatine kinase system plays an important role in the intracellular transport of energy from the mitochondria to the myofibrils and in the regulation of energy production coupled to energy utilization in this model system. Effective functional compartmentation of ATP could result from the binding of creatine kinase to cellular organelles (e.g., myofibrils and mitochondria) such that high energy charge at the myofibrils is maintained by the reverse creatine kinase reaction, while phosphocreatine is synthesized mainly at the mitochondria in the forward creatine kinase reaction. It was, therefore, essential to demonstrate the presence of mitochondrial creatine kinase in the cultured myocardial cells to support this hypothesis, particularly since the mitochondrial creatine kinase was reportedly absent in fetal hearts. Using electrophoresis on cellulose acetate strips, the mitochondrial creatine kinase isozyme, as well as MM, MB, and BB isozymes, have now been demonstrated in myocardial cultures derived from neonatal rats. The mitochondrial creatine kinase increased with age in culture and with age of animal from which the culture is derived. Furthermore, the addition of creatine to culture media stimulates its synthesis. The mitochondrial creatine kinase isozyme was not detected in nonmuscle cells in culture derived from the neonatal rat hearts, nor in L6 muscle cell line. Phosphocreatine was present in all cells, but the regulation of energy metabolism and energy shuttle by creatine-phosphocreatine–creatine kinase could be operative only in the cells where the mitochondrial creatine kinase is present. This regulatory mechanism provides for an efficient system concomitant with the continuous energy demand of the myocardium; it is not ubiquitous and its development in myocardial cells seems to be triggered postnatally.     

Creatine accumulation and exchange by HEK293 cells stably expressing high levels of a creatine transporter

We have generated a stable HEK293 cell line expressing high levels of a creatine transporter (CREAT). This cell line (HEK293-CREAT) was used to study the properties of CREAT in terms of the accumulation and release of creatine. HEK293-CREAT cells accumulated high steady state levels of creatine under saturating creatine levels (approx. 25-fold higher intracellular creatine levels than seen in control cells). The accumulation of high levels of creatine affected [3H]creatine uptake by decreasing the Vmax for transport. High intracellular creatine levels were maintained in the absence of extracellular creatine. External creatine stimulated the release of stored creatine by an exchange mechanism dependent on extracellular Na+. These studies have shown that cellular creatine levels can be affected by the amount of creatine transporter in the membrane and exchange through the creatine transporter. These findings highlight the importance of the creatine transporter in the maintenance of intracellular creatine levels.     

Cyclosporin A inhibits creatine uptake by altering surface expression of the creatine transporter

The immunosuppressive drug cyclosporin A (CsA) inhibited the hCRT-1 cDNA-induced creatine uptake in Xenopus oocytes and the endogenous creatine uptake in cultured C(2)C(12) muscle cells in a dose- and time-dependent manner. FK506, another potent immunosuppressant, was unable to mimic the effect of CsA suggesting that the inhibitory effect of CsA was specific. To delineate the mechanism underlying, we investigated the effect of CsA on the K(m) and V(max) of creatine transport and also on the cell surface distribution of the creatine transporter. Although CsA treatment did not affect the K(m) (20-24 microm) for creatine, it significantly decreased the V(max) of creatine uptake in both oocytes and muscle cells. CsA treatment reduced the cell surface expression level of the creatine transporter in the muscle cells by approximately 60% without significantly altering its total expression level, and the reduction in the cell surface expression paralleled the decrease in creatine uptake. Taken together, our results suggest that CsA inhibited creatine uptake by altering the surface abundance of the creatine transporter. We propose that CsA impairs the targeting of the creatine transporter by inhibiting the function of an associated cyclophilin, resulting in an apparent loss in surface expression of the creatine transporter. Our results also suggest that prolonged exposure to CsA may result in chronically creatine-depleted muscle, which may be a cause for the development of CsA-associated clinical myopathies in organ transplant patients.     

S-thiolation of cytoplasmic cardiac creatine kinase in heart cells treated with diamide

Two methods for quantitation of protein S-thiolation, by isoelectric focusing or by enzyme activity, were used for studying S-thiolation of cytoplasmic cardiac creatine kinase. With these methods, creatine kinase was identified as a major S-thiolated protein in both bovine and rat heart. In rat heart cell cultures, creatine kinase became 10% S-thiolated during a 10 min incubation with 0.2 mM diamide. This enzyme became S-thiolated more slowly than other heart cell proteins and it also dethiolated more slowly. Two sequential additions of diamide at a 25 min interval caused twice as much S-thiolation after the second addition as compared to the first. This increased sensitivity to the second diamide treatment may have resulted from glutathione loss during the first addition which produced a higher GSSG-to-GSH ratio after the second treatment. The GSSG-to-GSH ratio was highest prior to the maximum S-thiolation of creatine kinase, but, in general, the time course of glutathione was similar to the S-thiolation of creatine kinase. This study demonstrates that cytoplasmic creatine kinase is S-thiolated and, therefore, inhibited during a diamide-induced oxidative stress in heart cells. Implications for regulation of cardiac metabolism during oxidative stress are discussed.     

Electrolysis stimulates creatine transport and transporter cell surface expression in incubated mouse skeletal muscle: potential role of ROS

Electrical field stimulation of isolated, incubated rodent skeletal muscles is a frequently used model to study the effects of contractions on muscle metabolism. In this study, this model was used to investigate the effects of electrically stimulated contractions on creatine transport. Soleus and extensor digitorum longus muscles of male NMRI mice (35-50 g) were incubated in an oxygenated Krebs buffer between platinum electrodes. Muscles were exposed to [(14)C]creatine for 30 min after either 12 min of repeated tetanic isometric contractions (contractions) or electrical stimulation of only the buffer before incubation of the muscle (electrolysis). Electrolysis was also investigated in the presence of the reactive oxygen species (ROS) scavenging enzymes superoxide dismutase (SOD) and catalase. Both contractions and (to a lesser degree) electrolysis stimulated creatine transport severalfold over basal. The amount of electrolysis, but not contractile activity, induced (determined) creatine transport stimulation. Incubation with SOD and catalase at 100 and 200 U/ml decreased electrolysis-induced creatine transport by approximately 50 and approximately 100%, respectively. The electrolysis effects on creatine uptake were completely inhibited by beta-guanidino propionic acid, a competitive inhibitor of (creatine for) the creatine transporter (CRT), and were accompanied by increased cell surface expression of CRT. Muscle glucose transport was not affected by electrolysis. The present results indicate that electrical field stimulation of incubated mouse muscles, independently of contractions per se, stimulates creatine transport by a mechanism that depends on electrolysis-induced formation of ROS in the incubation buffer. The increased creatine uptake is paralleled by an increased cell surface expression of the creatine transporter.     

Effects of Amide Creatine Derivatives in Brain Hippocampal Slices, and Their Possible Usefulness for Curing Creatine Transporter Deficiency

The creatine/phosphocreatine system carries ATP from production to consumption sites and buffers the intracellular content of ATP at times of energy deprivation. The creatine transporter deficiency syndrome is an X-linked disease caused by a defective creatine transporter into the central nervous system. This disease is presently untreatable because creatine lacking its carrier cannot cross neither the blood–brain barrier nor the cell plasma membranes. Possible strategies to cure this condition are to couple creatine to molecules which have their own carrier, to exploit the latter to cross biological membranes or to modify the creatine molecule to make it more lipophilic, in such a way that it may more easily cross lipid-rich biological membranes. Such molecules could moreover be useful for treatment of stroke or other ischemic brain syndromes of normal (transporter working) tissue. In this paper we tested four molecules in in vitro hippocampal slices experiments to investigate whether or not they had a neuroprotective effect similar to that of creatine. On two of them we also performed biochemical measurements to investigate whether or not they were able to increase the creatine and phosphocreatine content of the hippocampal slices with and without block of the transporter. We found that these molecules increase levels of creatine after block of the transporter, and significantly increased the levels of phosphocreatine. Both significantly increased the total creatine content in both conditions of active and blocked transporter. This shows that these molecules are capable of entering cells through biological membranes without using the creatine transporter. By contrast, neither of them was able to delay synaptic block during anoxia of normal (transporter functioning) tissue. We conclude that these compounds might possibly be useful for therapy of creatine transporter deficiency, but further research is needed to understand their possible role in anoxia/ischemia of normal tissue.     

Roles of creatine in the regulation of cardiac protein synthesis

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.     

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.