Decreased Creatine Kinase Activity Caused by Electroconvulsive Shock

Although several advances have occurred over the past 20 years concerning the use and administration of electroconvulsive therapy to minimize side effects of this treatment, little progress has been made in understanding its mechanism of action. Creatine kinase is a crucial enzyme for brain energy homeostasis, and a decrease of its activity has been associated with neuronal death. This work was performed in order to evaluate creatine kinase activity from rat brain after acute and chronic electroconvulsive shock. Results showed an inhibition of creatine kinase activity in hippocampus, striatum and cortex, after acute and chronic electroconvulsive shock. Our findings demonstrated that creatine kinase activity is altered by electroconvulsive shock.     

Compartmentation of multiple forms of creatine kinase in the distal nephron of the rat kidney

Creatine kinase enzymes are present in tissues such as muscle and brain to interconvert creatine phosphate and ADP, thus providing a system to interconnect energy production and utilization (Bessman, S. P., and Carpenter, C. L. (1985) Annu. Rev. Biochem. 54, 831-862). Creatine kinase isoenzymes in kidney have received little attention since kidney contains relatively low creatine kinase activity compared with muscle and brain and because there is disagreement regarding the identity of the specific isoforms expressed in kidney. Using a combination of chromatographic and immunological techniques, we have identified two isoforms of creatine kinase in rat kidney supernatants, B creatine kinase, and the non-sarcomeric form of the mitochondrial creatine kinase, which represent 82 and 15%, respectively, of the total creatine kinase activity in this tissue. The identity of the non-muscle form of the mitochondrial creatine kinase was confirmed by N-terminal sequence analysis and compared with recently published cDNA sequences (Haas, R. C., and Strauss, A. W. (1990) J. Biol. Chem. 265, 6921-6927). We prepared multiple antisera specific for each isoform using synthetic peptide immunogens based upon nonhomologous regions from the primary sequence of each creatine kinase isoform. Immunocytochemical results demonstrate that both creatine kinase isoforms are colocalized in the inner stripe of the outer medulla in tubules of the distal nephron. A similar distribution of creatine kinase isoforms was obtained when different layers of the renal cortex and medulla were examined for creatine kinase activity and isozyme content using nondenaturing electrophoresis. In general, the distribution of creatine kinase enzymes in kidney corresponds to the regions of greatest ATP utilization, oxygen consumption, and sodium transport. These results suggest a role for creatine kinase enzymes in the coupling of ion transport and oxidative phosphorylation in the distal nephron of the mammalian kidney.     

Creatine induction of creatine kinase synthesis in a developing monolayer culture of muscle cells]

Creatine action on the activity of creatine kinase (ATP: creatine-phosphotransferase; EC 2.7.3.2) and the content of water-soluble proteins in the developing monolayer culture of chick myoblasts are studied. Creatine at concentrations of 1.9-10- minus 3-3.8-10- minus 3 M is shown to increase reliably the creatine kinase activity by 1,1--2,9 times and to reduct considerably the content of water-soluble proteins. Lower concentrations of creatine (3.8-10- minus 5 M) also increased the creatine kinase activity but did not change the contents of water-soluble proteins. The creatine effect was maximal at the period preceding the termination of tissue cells differentiation. In the course of the combined effect of both actinomycin D (50 mcg/plate) and creatine (3.8-10- minus 3 M) the creatine kinase activity was much higher than that in the presence of actinomycin D alone which considerably reduced the enzyme activity as well as the contents of water-soluble proteins.     

Developmental studies on creatine kinase. Isoenzyme in rat gastrointestinal tract

Creatine kinase activity and its isoenzymatic profile in rat intestinal mucose during normal development have been studied. Creatine kinase enzymatic activity increased stepwise during fetal development and the first week of life. An isoenzymatic pattern of exclusively CK-BB types occurred in all segments of the digestive tract during the early fetal stage. The isoenzyme profile of creatine kinase in the esophagic tissue with advancing maturation of the fetus shifted in the same way as in adults, with preferential concentration of CK-MM. However, CK-BB continued to be the main isoenzyme in the rest of the digestive tract. Our results show that rats are particularly suitable for experimental studies of intestinal creatine kinase isoenzymes.     

Evidence against regulation of AMP-activated protein kinase and LKB1/STRAD/MO25 activity by creatine phosphate

Muscle contraction results in phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an AMPK kinase (AMPKK). LKB1/STRAD/MO25 (LKB1) is the major AMPKK in skeletal muscle; however, the activity of LKB1 is not increased by muscle contraction. This finding suggests that phosphorylation of AMPK by LKB1 is regulated by allosteric mechanisms. Creatine phosphate is depleted during skeletal muscle contraction to replenish ATP. Thus the concentration of creatine phosphate is an indicator of cellular energy status. A previous report found that creatine phosphate inhibits AMPK activity. The purpose of this study was to determine whether creatine phosphate would inhibit 1) phosphorylation of AMPK by LKB1 and 2) AMPK activity after phosphorylation by LKB1. We found that creatine phosphate did not inhibit phosphorylation of either recombinant or purified rat liver AMPK by LKB1. We also found that creatine phosphate did not inhibit 1) active recombinant alpha1beta1gamma1 or alpha2beta2gamma2 AMPK, 2) AMPK immunoprecipitated from rat liver extracts by either the alpha1 or alpha2 subunit, or 3) AMPK chromatographically purified from rat liver. Inhibition of skeletal muscle AMPK by creatine phosphate was greatly reduced or eliminated with increased AMPK purity. In conclusion, these results suggest that creatine phosphate is not a direct regulator of LKB1 or AMPK activity. Creatine phosphate may indirectly modulate AMPK activity by replenishing ATP at the onset of muscle contraction.     

Mitochondrial respiratory chain and creatine kinase activities in rat brain after sepsis induced by cecal ligation and perforation

The mechanisms responsible to the development of brain dysfunction during sepsis are not well understood. The objective of this study is to evaluate mitochondrial respiratory chain and creatine kinase activities in the brain after cecal ligation and perforation (CLP) in rats. We performed a prospective, controlled experiment in male Wistar rats. Rats were subjected to CLP (sepsis group) with saline resuscitation (at 50mL/kg immediately and 12h after cecal ligation and perforation) or sham operation (control group). Several times (0, 6, 12, 24, 48 and 96h) after CLP six rats were killed by decapitation, and brain structures (cerebellum, hippocampus, striatum and cortex) were isolated. Mitochondrial respiratory chain and creatine kinase activity were then measured. It was observed that animals submitted to CLP presented decreased mitochondrial respiratory chain activity in complex I, but not in complex II, III and IV, 24, 48 and 96h in all analyzed structures. Activity of succinate dehydrogenase was decreased in 48 and 96h in all analyzed structures. Creatine kinase activity increased after CLP in cerebellum, hippocampus and cortex (after 0h) and striatum (after 6h). Sepsis associated brain injury may include dysfunction in the mitochondrial respiratory chain activity.     

Identification of a 43-kDa polypeptide associated with acetylcholine receptor-enriched membranes as MM creatine kinase

Creatine kinase isoenzymes from Torpedo californica electric organ, skeletal muscle, and brain were purified and characterized. Torpedo electric organ and skeletal muscle creatine kinase have identical apparent Mr, electrophoretic mobility, and cyanogen bromide fragments. The electrophoretic mobility of the Torpedo creatine kinase was anodal as compared to mammalian MM creatine kinase. No creatine kinase isoenzyme with an electrophoretic mobility similar to mammalian BB creatine kinase was seen in any of the Torpedo tissues examined. Hybridization studies demonstrate the Torpedo electric organ creatine kinase to be composed of identical subunits and capable of producing an enzymatically active heterodimer when combined with canine BB creatine kinase. Creatine kinase from sucrose gradient-purified Torpedo electric organ acetylcholine receptor-rich membranes has an electrophoretic mobility identical with the cytoplasmic isoenzyme and an apparent Mr identical with mammalian MM creatine kinase. Western blot analysis showed Torpedo electric organ skeletal muscle creatine kinase and acetylcholine receptor-enriched membrane creatine kinase reacted with antiserum specific for canine MM creatine kinase. NH2-terminal amino acid sequence determinations show considerable sequence homology between human MM, Torpedo electric organ, chicken MM, and porcine MM creatine kinase. The acetylcholine receptor-associated creatine kinase is, therefore, identical with the cytoplasmic form from the electric organ and is composed of M-subunits.     

Mitochondrial component of the phosphorylcreatine shuttle is enhanced during rat heart perinatal development

Aerobic metabolism is enhanced during perinatal heart development in parallel with increased cardiac function. The mitochondrial component of the phosphorylcreatine shuttle is important in providing energy for contraction and was examined in weanling and adult rat left ventricle. Creatine kinase activity was enhanced in tissue homogenate and purified cardiac myocytes of adults. Mitochondrial analyses attribute this enhancement to increased creatine kinase activity per milligram mitochondrial protein. Other enzymatic markers of mitochondrial function are not enhanced in activity during perinatal heart growth. The unique response of creatine kinase points to the shuttle mechanism and of mitochondrial creatine kinase, in particular, as a major contributor to heart functional regulation.     

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.     

CREATINE KINASE FROM BRAIN: KINETIC ASPECTS

Abstract— Creatine kinase derived from rabbit brain has been re-examined with respect to its kinetic features. The enzyme from brain has lower Michaelis constants for both ADP and creatine phosphate than does the enzyme from rabbit muscle. Substrate inhibition by excess creatine phosphate occurs at a concentration approximating that found in the tissue. The enzyme from muscle is less sensitive to substrate inhibition.
The crude mitochondrial fraction from rat brain was centrifuged in a sucrose density gradient and the distribution of enzymatic activities among the subfractions was determined. The distribution of creatine kinase resembled that of two glycolytic enzymes; no evidence for a mitochondrial localization was found.     

Neuroprotective effects of creatine

There is a substantial body of literature, which has demonstrated that creatine has neuroprotective effects both in vitro and in vivo. Creatine can protect against excitotoxicity as well as against β-amyloid toxicity in vitro. We carried out studies examining the efficacy of creatine as a neuroprotective agent in vivo. We demonstrated that creatine can protect against excitotoxic lesions produced by N-methyl-d-aspartate. We also showed that creatine is neuroprotective against lesions produced by the toxins malonate and 3-nitropropionic acid (3-NP) which are reversible and irreversible inhibitors of succinate dehydrogenase, respectively. Creatine produced dose-dependent neuroprotective effects against MPTP toxicity reducing the loss of dopamine within the striatum and the loss of dopaminergic neurons in the substantia nigra. We carried out a number of studies of the neuroprotective effects of creatine in transgenic mouse models of neurodegenerative diseases. We demonstrated that creatine produced an extension of survival, improved motor performance, and a reduction in loss of motor neurons in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). Creatine produced an extension of survival, as well as improved motor function, and a reduction in striatal atrophy in the R6/2 and the N-171-82Q transgenic mouse models of Huntington’s disease (HD), even when its administration was delayed until the onset of disease symptoms. We recently examined the neuroprotective effects of a combination of coenzyme Q10 (CoQ10) with creatine against both MPTP and 3-NP toxicity. We found that the combination of CoQ and creatine together produced additive neuroprotective effects in a chronic MPTP model, and it blocked the development of alpha-synuclein aggregates. In the 3-NP model of HD, CoQ and creatine produced additive neuroprotective effects against the size of the striatal lesions. In the R6/2 transgenic mouse model of HD, the combination of CoQ and creatine produced additive effects on improving survival. Creatine may stabilize mitochondrial creatine kinase, and prevent activation of the mitochondrial permeability transition. Creatine, however, was still neuroprotective in mice, which were deficient in mitochondrial creatine kinase. Administration of creatine increases the brain levels of creatine and phosphocreatine. Due to its neuroprotective effects, creatine is now in clinical trials for the treatment of Parkinson’s disease (PD) and HD. A phase 2 futility trial in PD showed approximately a 50% improvement in Unified Parkinson’s Disease Rating Scale at one year, and the compound was judged to be non futile. Creatine is now in a phase III clinical trial being carried out by the NET PD consortium. Creatine reduced plasma levels of 8-hydroxy-2-deoxyguanosine in HD patients phase II trial and was well-tolerated. Creatine is now being studied in a phase III clinical trial in HD, the CREST trial. Creatine, therefore, shows great promise in the treatment of a variety of neurodegenerative diseases.     

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.

     

Creatine kinase BB in normal, hyperplastic and neoplastic endometrium

We measured creatine kinase BB activity in normal, hyperplastic and neoplastic endometrium. Creatine kinase BB levels were higher in secretory than in proliferative endometrium. High levels were also found in hyperplastic tissue. In endometrial cancer creatine kinase BB activity was higher in well differentiated than in poorly differentiated tumors.     

Creatine kinase isoenzymes in Torpedo californica: absence of the major brain isoenzyme from nicotinic acetylcholine receptor membranes

Creatine kinase, actin, and nu 1 are three proteins of Mr 43 000 associated with membranes from electric organ highly enriched in nicotinic acetylcholine receptor. High levels of creatine kinase are required to maintain adequate ATP levels, while actin may play a role in maintaining the synaptic cytoskeleton. Previous investigations have prompted the conclusion that postsynaptic specializations at the receptor-enriched membrane domains in electroplax contain the brain form of creatine kinase rather than the form of creatine kinase predominantly found in muscle. We have examined this conclusion by purifying Torpedo brain creatine kinase to virtual homogeneity in order to examine its immunochemical, molecular, and electrophoretic properties. On the basis of immunological cross-reactivity and isozyme analysis, the receptor-associated creatine kinase is identified to be of the muscle type. When the molecular characteristics of Torpedo brain and muscle creatine kinase are compared, the brain enzyme is positioned at a more basic pH during chromatofocusing and on two-dimensional gel electrophoresis (pI = 7.5-7.9). Furthermore, electrophoretic mobilities of the brain and muscle forms of creatine kinase differ in sodium dodecyl sulfate electrophoresis: the brain isozyme of creatine kinase has lower apparent molecular weight (Mr 41 000) when compared with the muscle enzyme (Mr 43 000). On the basis of the results of our current investigations, the hypothesis that the brain isozyme of creatine kinase is a component of the postsynaptic specializations of the Torpedo californica electroplax must be abandoned. Recent sequence data have established close homology between Torpedo and mammalian muscle creatine kinases. On the basis of electrophoretic criteria, our results indicate that a lower degree of homology exists between the brain isozymes.     

Purification and characterization of creatine kinase isozymes from the nurse shark Ginglymostoma cirratum

Creatine kinase from nurse shark brain and muscle has been purified to apparent homogeneity. In contrast to creatine kinases from most other vertebrate species, the muscle isozyme and the brain isozyme from nurse shark migrate closely in electrophoresis and, unusually, the muscle isozyme is anodal to the brain isozyme. The isoelectric points are 5.3 and 6.2 for the muscle and brain isozymes, respectively. The purified brain preparation also contains a second active protein with pI 6.0. The amino acid content of the muscle isozyme is compared with other isozymes of creatine kinase using the Metzger Difference Index as an estimation of compositional relatedness. All comparisons show a high degree of compositional similarity including arginine kinase from lobster muscle. The muscle isozyme is marginally more resistant to temperature inactivation than the brain isozyme; the muscle protein does not exhibit unusual stability towards high concentrations of urea. Kinetic analysis of the muscle isozyme reveals Michaelis constants of 1.6 mM MgATP, 12 mM creatine, 1.2 mM MgADP and 50 mM creatine phosphate. Dissociation constants for the same substrate from the binary and ternary enzyme-substrate complex do not differ significantly, indicating limited cooperatively in substrate binding. Enzyme activity is inhibited by small planar anions, most severely by nitrate. Shark muscle creatine kinase hybridizes in vitro with rabbit muscle or monkey brain creatine kinase; shark brain isozyme hybridizes with monkey brain or rabbit brain creatine kinase. Shark muscle and shark brain isozymes, under a wide range of conditions, failed to produce a detectable hybrid.     

Contact site between inner and outer mitochondrial membrane: a dynamic microcompartment for creatine kinase activity

Creatine kinase is involved in the integration of high-energy metabolism in various tissues. In this study the tissue-specific distribution of the mitochondrial isoform was investigated, both by electrophoresis of rat tissue extracts, and by ultrastructural localisation of creatine kinase activity. Furthermore, the influence of uncoupling of oxidative phosphorylation on mitochondrial creatine kinase activity associated with intermembrane contacts was investigated by enzyme cytochemistry and morphometric analysis. The results of the cytochemical survey indicate that contact sites are a prerequisite for creatine kinase to demonstrate enzymatic activity. Moreover, the extent of creatine kinase active membrane contacts depends on the metabolic state of the mitochondrion, as shown for heart mitochondria in vivo and in vitro, before and after treatment with dinitrophenol.     

Biology of reproduction in the squirrel monkey (Saimiri sciureus): II. The ovary

Gross and microscopic observations on the squirrel monkey ovary is described. The luteal tissues are present in two broadly distinct stages of activity. The degree of follicular development appears to be affected by the level of luteal activity. Circumstantial evidences have been presented to indicate that squirrel monkey females may be an induced ovulator.Supported by Grant HDO 1952–03 from the United States Public Health Service.     

Creatine kinase of rat lymphocytes and its activity as affected by adenosine, cAMP, AMP and products of their transformation

Creatine kinase is found in the thymus and spleen lymphocytes of rats. Its activity in the spleen lymphocytes is considerably higher than in the thymocytes. The data of enzyme-electrophoresis chromatography on DEAE-Sephadex A-50, thermostability and pH optimum indicate that lymphocyte creatine kinase is a "cerebral" isoenzyme. It is mainly concentrated in hyaloplasm and mitochondrial fraction and, to a less extent--in the microsomal and nuclear-cellular fractions. It is shown that adenosine, inosine, hypoxanthin, NH4, cAMP and GTP have no effect on the creatine kinase activity of thymocytes but pyrophosphate causes its decrease. Creatine kinase of lymphocytes as compared with its myocardium isoenzyme MM possesses a less substrate specificity to nucleotide diphosphates.     

Studies on the mitochondrially bound form of rat brain creatine kinase

1. The development of the total rat brain creatine kinase was studied in brain homogenates. Until approx. 14-15 days after birth, the activity remains less than one-third that of the adult activity (207+/-6 units/g wet wt. s.d.; n=3). Over the next 10 days the activity increases markedly to the adult value and thereafter remains essentially constant. 2. In the adult brain, approx. 5% (11.9+/-2.2 units/g wet wt. s.d.; n=5) of the total creatine kinase is associated with the mitochondrial fraction. This creatine kinase could not be solubilized by sodium acetate solutions of up to 0.8m concentration, whereas 66% of the hexokinase associated with brain mitochondria was released under these conditions. 3. Rat brain mitochondria incubated in the presence of various concentrations of creatine (1, 5 and 10mm) and ADP (100mum) synthesized phosphocreatine at rates of approx. 4.5, 11 and 17.5nmol/min per mg of mitochondrial protein. Atractyloside (50mum) or oligomycin (1.5mug/mg of mitochondrial protein) completely inhibited the synthesis of phosphocreatine. 4. The apparent K(m) and V(max.) values of the mitochondrially bound rat brain creatine kinase were determined in both directions. The V(max.) in the direction of phosphocreatine synthesis is 237nmol/min per mg of mitochondrial protein, with an apparent K(m) for creatine of 1.67mm and for MgATP(2-) of 0.1mm, and in the reverse direction V(max.) is 489nmol/min per mg of mitochondrial protein, with an apparent K(m) for phosphocreatine of 0.4mm and for MgADP(-) of 27mum. 5. The results are discussed with reference to the role that the mitochondrially bound creatine kinase may play in the development of brain energy metabolism.     

Overexpression, purification, and preliminary X-ray crystallographic analysis of human brain-type creatine kinase

Creatine kinase (CK; E.C. 2.7.3.2) is an important enzyme that catalyzes the reversible transfer of a phosphoryl group from ATP to creatine in energy homeostasis. The brain-type cytosolic isoform of creatine kinase (BB-CK), which is found mainly in the brain and retina, is a key enzyme in brain energy metabolism, because high-energy phosphates are transferred through the creatine kinase/phosphocreatine shuttle system. The recombinant human BB-CK protein was overexpressed as a soluble form in Escherichia coli and crystallized at 22 degrees C using PEG 4000 as a precipitant. Native X-ray diffraction data were collected to 2.2 A resolution using synchrotron radiation. The crystals belonged to the tetragonal space group P43212, with cell parameters of a=b=97.963, c= 164.312 A, and alpha=beta=gamma=90 degrees. The asymmetric unit contained two molecules of CK, giving a crystal volume per protein mass (Vm) of 1.80 A3 Da-1 and a solvent content of 31.6%.