Effect of calcium on creatine kinase activity of cerebellum

Summary The effect of Ca⁺⁺ ions on the histochemical activity of creatine kinase (CK) was investigated in striated muscle and cerebellum of the rat. The intensity and pattern of CK activity was unchanged in the striated muscle when Ca⁺⁺ was present in the incubation medium instead of Mg⁺⁺. In the cerebellum, however, Ca⁺⁺ inhibited the enzymatic activity except in the Purkinje cells.     

Effect of calcium on creatine kinase activity of cerebellum.

The effect of Ca++ ions on the histochemical activity of creatine kinase (CK) was investigated in striated muscle and cerebellum of the rat. The intensity and pattern of CK activity was unchanged in the striated muscle when Ca++ was present in the incubation medium instead of Mg++. In the cerebellum, however, Ca++ inhibited the enzymatic activity except in the Purkinje cells.     

Hydroxyurea inhibits thymidine kinase activity in developing rat cerebellum

Hydroxyurea when injected intraperitoneally into rats either as a single dose or as three consecutive daily doses, markedly inhibited thymidine kinase activity in cerebellum on 7th day. The inhibitory effect of the drug was found to be both dose and time dependent. The drug has however, failed to exert any inhibitory action when added to the reaction mixture in vitro. It is concluded that the well established inhibition on DNA synthesis by hydroxyurea may not be solely due to its action on ribonucleotide reductase (EC 1.17.4.1) but probably due to its interference at several other sites including thymidine kinase.     

Inhibition of mitochondrial creatine kinase activity by D-2-hydroxyglutaric acid in cerebellum of young rats

D-2-Hydroxyglutaric aciduria (DHGA) is a neurometabolic disorder biochemically characterized by tissue accumulation and excretion of high amounts of D-2-hydroxyglutaric acid (DGA). Although the affected patients have predominantly severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In previous studies we have demonstrated that DGA, at concentrations as low as 0.25 mM, significantly decreased creatine kinase activity and other parameters of energy metabolism in cerebral cortex of young rats. In the present study, we investigated the effect of DGA (0.25-5 mM) on total creatine kinase (tCK) activity, as well as on CK activity in cytosolic (Cy-CK) and mitochondrial (Mi-CK) preparations from cerebellum of 30-day-old Wistar rats in order to test whether the inhibitory effect of DGA on CK was tissue specific. We verified that tCK (22% inhibition) and Mi-CK (40% inhibition) activities were moderately inhibited by DGA at concentrations of 2.5 mM and higher, in contrast to Cy-CK, which was not affected by the acid. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by preincubation of the homogenates with reduced glutathione, suggesting that the inhibition of CK activity by DGA is possibly mediated by modification of essential thiol groups of the enzyme. Our present results therefore demonstrate a relatively weak inhibitory effect of DGA on cerebellum Mi-CK activity, as compared to that provoked in cerebral cortex, and may possibly be related to the neuropathology of DHGA, characterized by cerebral cortex abnormalities.     

Acute changes of myocardial creatine kinase gene expression under beta-adrenergic stimulation

Creatine kinase (CK) plays a crucial role in myocardial energy metabolism. Alterations in CK gene expression are found in hypertrophied and failing heart, but the mechanisms behind these changes are unclear. This study tests the hypothesis that increased adrenergic stimulation, which is observed in heart failure, induces changes of myocardial CK-activity, -isoenzyme distribution and -gene expression that are characteristic of the failing and hypertrophied heart. Isolated rat hearts were perfused (constant pressure of 80 mmHg) with red cell suspensions. Following a 20-min warm-up period, perfusion for 3 h with 10(-8) M (iso 3 h) or without (control 3 h) isoproterenol was started or experiments were immediately terminated (control 0 h). Left ventricular tissue was analyzed for total CK-activity, CK-isoenzyme distribution and, by use of quantitative RT-PCR, for B-CK, M-CK, mito-CK and GAPDH- (as internal standard) mRNA. After beta-adrenergic stimulation (iso 3 h) but not after control perfusion (control 3 h) a roughly threefold increase in B-CK mRNA levels and a decrease in M-CK mRNA levels by 18% was found. There were no significant differences among the three groups in total CK-activity and in distribution of CK-MM, CK-BB, CK-MB and mito-CK. Thus, beta-adrenergic stimulation induces a switch in CK gene expression from M-CK to B-CK, which is characteristic for the hypertrophied and failing heart. This may be interpreted as an adaptive mechanism making energy transduction via CK more efficient at times of increased metabolic demand.     

Effect of vitamin D and its metabolites on developing myogenic cultures

Growth, protein synthesis and expression of creatine kinase (CK) by embryonic chick myogenic cells are inhibited by vitamin D and certain of its metabolites. 25-OH cholecalciferol was most active in concentrations of 10⁻⁵–10⁻⁶ M, with cholecalciferol and ergocalciferol less active in that order. Ergosterol had no activity of this sort. Inhibition of CK was most marked on the 4th and 5th day of culture and was due to suppression of the appearance of CK-MM and MB. CK-BB was not affected and CK-MB was more affected than CK-BB. Skin fibroblasts by comparison were slightly stimulated in growth at 10⁻⁶ M and much less affected at 10⁻⁵ M than the myogenic cells. It is suggested that vitamin D has a direct effect upon the muscle cell, to cause a selective diminution in the production of certain polypeptides.     

Myofibrillar creatine kinase activity inferred from a 3D model

Myofibrillar creatine kinase (CK) that buffers ATP during fluctuating muscle energy metabolism has been selected for studies of conformational changes underlying the cellular control of enzyme activity. The force field was computed for three energetic states, namely for the substrate-free CK molecule, for the molecule conjugated with the MgATP complex, and for the molecule conjugated with the pair of reactants MgATP-creatine. Without its substrates, the enzyme molecule assumes an inactive "open" form. Upon binding of the MgATP complex, the CK molecule takes up a reactive "closed" conformation. Subsequent binding of creatine yields a nonreactive "intermediary" conformation. Acid-base catalysis is considered to be the basic principle for the reversible transfer of the phosphoryl group between the substrates. The results indicate that the substrate-induced energy minimizing conformational changes do not represent a sufficient condition for CK activity and that some other essential component of physiological control at the cellular level is involved in the transition from the intermediary to the closed structure of the molecule.     

Modulation of creatine kinase activity by ruthenium complexes

Creatine kinase is a crucial enzyme for brain, heart and skeletal muscle energy homeostasis, and a decrease of its activity has been associated with cell death. Many biological properties have been attributed to ruthenium complexes. In this context, this work was performed in order to evaluate creatine kinase activity from rat brain, heart and skeletal muscle (quadriceps) after administration of ruthenium complexes, trans-[RuCl(2)(nic)(4)] (nic=3-pyridinecarboxylic acid) 180.7 micromol/kg (complex I), trans-[RuCl(2)(i-nic)(4)] (i-nic=4-pyridinecarboxylic acid) 13.6 micromol/kg (complex II), trans-[RuCl(2)(dinic)(4)] (dinic=3,5-pyridinedicarboxylic acid) 180.7 micromol/kg (complex III) and trans-[RuCl(2)(i-dinic)(4)] (i-dinic=3,4-pyridinedicarboxylic acid) 180.7 micromol/kg (complex IV). Our results showed that complex I caused inhibition of creatine kinase activity in hippocampus, striatum, cerebral cortex, heart and skeletal muscle. Besides, complex II did not affect the enzyme activity. complexes III and IV increased creatine kinase activity in hippocampus, striatum, cerebral cortex and heart, but not in skeletal muscle. Besides, none of the complexes in vitro altered creatine kinase activity, suggesting that enzymatic activity is indirectly affected by complexes I, III and IV. It is believed that diminution of creatine kinase in brain of rats caused by complex I may be related to results from other study reporting memory impairment caused by the same complex. Further research is necessary in order to elucidate the effects of ruthenium complexes in other important metabolic enzymes.     

Support of embryonic chick survival by vitamin D metabolites

The provision of 1,25-dihydroxyvitamin D3 as the only source of dietary vitamin D3 to laying hens failed to support normal embryonic development in their fertile eggs. Significant (P less than .001) improvement in embryonic survival to hatching in these eggs resulted from injections of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or 24,24-difluoro-25-hydroxyvitamin D3 prior to incubation. Maximum embryonic survival with lowest embryonic mortality was observed when 0.20 micrograms/egg of 1,25-dihydroxyvitamin D3 or 0.60 micrograms/egg 25-hydroxyvitamin D3 was injected. These results indicate that several forms of vitamin D, two of which cannot be converted to 24,25-dihydroxyvitamin D3, can provide this activity; and of the vitamin D compounds tested, 1,25-dihydroxyvitamin D3 may be the most active in supporting embryonic survival in the chick when delivered directly by injection.     

Regulation of creatine phosphokinase B activity by protein kinase C

We previously reported that topical application of 12-o-tetradecanoylphorbol-13-acetate to mouse skin causes phosphorylation of epidermal proteins with molecular weights of 40,000 (p40) and 34,000 (p34). In the accompanying paper, p40 was identified as creatine phosphokinase B. Here we report that both in intact cells and in a cell-free system, phosphorylation of creatine hosphokinase B by protein kinase C resulted in an increase in its ability to catalyze the transfer of the high-energy phosphate of phosphocreatine to ADP, thereby producing ATP. H-7, a specific inhibitor of protein kinase C was found to abolish the increase in enzyme activity. Lineweaver-Burk plot analysis indicated that the increased activity was mostly due to a decreased Km for phosphocreatine. Phosphorylation and activation of creatine phosphokinase B may be a physiological response to maintain ATP balance when a protein kinase C pathway is stimulated.     

Physiological role of the creatine kinase system and the problem of regulating the activity of mitochondrial creatine kinase

The review contains the analysis of present-day concepts on the physiological role of the creatine kinase system. A hypothesis on the buffering functions of the creatine kinase system which ensures a constant ATP level in cells and a hypothesis according to which phosphocreatine is a macroergic phosphate carrier from mitochondria to the sites of their utilization are considered. In connection with the creatine phosphate carrier hypothesis according to which the transport function of the creatine kinase system is provided for by an effective function of mitochondrial creatine kinase, feasible mechanisms of mitochondrial creatine kinase activity regulation are considered: as a result of creation of local concentration of nucleotide substrates as well as changes in the properties of creatine kinase itself which may result from the enzyme conversion from the membrane-bound to the free form or from the interconversion of oligomeric forms of the enzyme.     

Nongenomic regulation of extracellular matrix events by vitamin D metabolites

Vitamin D metabolites appear to regulate chondrocytes and osteoblasts via a combination of genomic and nongenomic mechanisms. Specificity of the nongenomic response to either 1,25-(OH)₂D₃ or 24, 25-(OH)₂D₃ may be conferred by the chemical composition of the target membrane and its fluid mosaic structure, by the presence of specific membrane receptors, or by the interaction with classic Vitamin D receptors. Nongenomic effects have been shown to include changes in membrane fluidity, fatty acid acylation and reacylation, arachidonic acid metabolism and prostaglandin production, calcium ion flux, and protein kinaase C activity. Chondrocytes metabolize 25-(OH)D₃ to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃; production of these metabolites is regulated by both growth factors and hormones and is dependent on the state of cell maturation. 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ may interact directly with extracellular matix vesicles to regulate their function in the matrix, including protease activity, resulting in matrix modefication and calcification. Isolated matrix vesicles, produced by growth zone chondrocytes, can activate latent transforming growth factor-β when incubated with exogenous 1,25-(OH)₂D₃. These observations suggest that nongenomic regulation of martix vesicle structure and function may be a mechanism by which mesenchymal cells, like osteoblasts and chndrocytes, may modulate events in the extracellular matrix at sites distant from the cell surace.     

Modulation of myelomonocytic U937 cells by vitamin D metabolites

Investigation of the effects of 1,25(OH)₂D₃ and 24,25(OH)₂D₃ on the proliferation and differentiation of the human myelomonocytic cell line U937 has been complemented with studies of the effect of the same metabolites on the number of nuclear receptors for 1,25(OH)₂D₃. Both 1,25(OH)₂D₃ and 24,25(OH)₂D₃ inhibit the proliferation of U937 cells in a dose-dependent manner. The concentrations of 24,25(OH)₂D₃ required to produce this effect were 100-times greater than those of 1,25(OH)₂D₃. Inhibition of proliferation was associated with increased expression of the CD14 and 200 kDa 63D3 antigens thus confirming differentiation of U937 towards a more mature cell type.Studies of the nuclear receptor for 1,25(OH)₂D₃ showed that pre-treatment of the cells with 1,25(OH)₂D₃ resulted in an apparent 40% decrease in the number of detectable 1,25(OH)₂D₃ receptors as compared to control U937 cells. This is due to the fact that the 1,25(OH)₂D₃ binds to U937 cell nuclei during culture and thus blocks the subsequent binding of radiolabelled 1,25(OH)₂D₃ used to measure the number of 1,25(OH)₂D₃ receptors. Measurement of the binding of unlabelled 1,25(OH)₂D₃ by radioimmunoassay indicated that pre-treatment of the cells with 1,25(OH)₂D₃ increased the capacity of U937 to bind the hormone, although measurement of these receptors by whole cell assay was prevented by the binding of 1,25(OH)₂D₃ itself. This effect was not observed with 24,25(OH)₂D₃ which was more easily displaced from binding sites by radiolabelled 1,25(OH)₂D₃ and it appears to act through low affinity binding to the 1,25(OH)₂D₃ receptor.     

Imaging of human brain creatine kinase activity in vivo

Creatine kinase activity and high-energy phosphate concentration have been investigated using localized 31P spectroscopy in the human brain in vivo. The phase-modulated rotating frame imaging technique, incorporating magnetization transfer and inversion recovery, has been used to produce a 1-dimensional rate profile map of steady-state enzyme activity. Large differences in the flux from phosphocreatine (PCr) to ATP have been discovered between volumes of human brain consisting of predominantly gray (2.0 cm) and white (4.5 cm) matter. The concentration of PCr changes slightly (2.0 cm = 5.20 +/- 0.45 mmol.l-1, 4.5 cm = 4.63 +/- 0.31 mmol.l-1), while the ATP concentration remains within limits (3.30 +/- 0.4 mmol.l-1). No change in pHi was detected between the two regions in normal volunteers (n = 6). The forward rate constant of the PCr----ATP reaction in regions of predominantly gray matter (0.30 +/- 0.04 s-1) was twice that of white matter (0.16 +/- 0.02 s-1) in vivo.     

Occurrence of creatine kinase activity in human erythrocyte membrane

Some evidences for creatine kinase activity in normal human erythrocyte membrane were presented. The creatine kinase was indicated to be a constituent of the integral proteins of erythrocyte membrane or to be tightly bound to the membrane, and was contrasted to the results obtained with adenylate kinase. Isoenzyme distribution of the erythrocyte creatine kinase by electrophoresis was identical to MM-creatine kinase from rabbit muscle.