Extracellular ATP (exATP) has been known to be a critical ligand regulating skeletal muscle differentiation and contractibility. ExATP synthesis was greatly increased with the high level of adenylate kinase 1 (AK1) and ATP synthase beta during C2C12 myogenesis. The exATP synthesis was abolished by the knock-down of AK1 but not by that of ATP synthase beta in C2C12 myotubes, suggesting that AK1 is required for exATP synthesis in myotubes. However, membrane-bound AK1beta was not involved in exATP synthesis because its expression level was decreased during myogenesis in spite of its localization in the lipid rafts that contain various kinds of receptors and mediate cell signal transduction, cell migration, and differentiation. Interestingly, cytoplasmic AK1 was secreted from C2C12 myotubes but not from C2C12 myoblasts. Taken together all these data, we can conclude that AK1 secretion is required for the exATP generation in myotubes.
Adenosine Triphosphate/*biosynthesis ; Adenylate Kinase/*metabolism ; Animals ; Cell Line ; Extracellular Space/metabolism ; Isoenzymes/*metabolism ; Mice ; Muscles/cytology/*metabolism

Proteolytic degradation of the extracellular matrix and tumor metastasis correlate with the expression of endopeptidases known as matrix metalloproteinases (MMPs). Expression of MMPs is regulated by cytokines and signal transduction pathways including those activated by phorbol myristate acetate (PMA). We found that resveratrol, a phytoalexin present in grapes, significantly inhibits the PMA-induced increase of MMP-9 expression and activity. These effects of resveratrol were dose-dependent and correlated with the suppression of MMP-9 mRNA expression levels. PMA caused a 23-fold increase in MMP-9 promoter activity, which was suppressed by resveratrol. Transient transfection by MMP-9 constructs, in which specific transcriptional factors were mutagenized, indicated that the effects of PMA and resveratrol were mediated via AP1 and NFkB response elements. Resveratrol inhibited PMA-mediated activation of c-Jun Nterminal kinase (JNK) and protein kinase C (PKC)-delta activation. Therefore, we conclude that the inhibitory activities of resveratrol on MMP-9, JNK, and PKC-delta may have therapeutic potential for controlling growth and invasiveness of tumors.
Cytokines ; Endopeptidases ; Extracellular Matrix ; Matrix Metalloproteinases ; Neoplasm Metastasis* ; Phosphotransferases ; Protein Kinase C ; Response Elements ; RNA, Messenger ; Signal Transduction ; Tetradecanoylphorbol Acetate ; Transfection ; Vitis

In comparison with its counterpart from N. meningitides, all conserved motifs were found in the N-termini of E. coli CMP-NeuAc synthetase. E. coli CMP-NeuAc synthetase seems to have redundant C-termini with a less effect on its activity. To explain this speculation, a series of recombinant DNAs with deletion from 3'-end of CMP-NeuAc synthetase were produced by PCR, ligated into expression vector pET-15b and expressed in BL21(DE3)pLysS. After induction with IPTG, we found that the recombinant enzyme with deletion of 189 amino acids from C0termini retained its activity. This result demonstrates that the 229 amino acids of N-termini was the minimal functional domain of E. coli CMP-NeuAc synthetase. The deletions altered the optimum pH and thermostability of active truncated enzymes, indicating that the truncated C-terminal amino acids of E. coli CMP-NeuAc synthetase could affect the conformation of the enzymatic catalytic domain and therefore affect its catalytic activity and thermostability, although it is not involved in enzymatic activity directly.
Amino Acid Sequence ; Cytidine Monophosphate N-Acetylneuraminic Acid ; metabolism ; Enzyme Stability ; Escherichia coli ; enzymology ; Hydrogen-Ion Concentration ; Molecular Sequence Data ; Mutation ; N-Acylneuraminate Cytidylyltransferase ; chemistry ; physiology ; Structure-Activity Relationship

BACKGROUND: Recent studies suggest that the cardioprotective effect of ischemic preconditioning (IPC) is related to intracellular glycogen content in rat hearts, however, controversies still remain. METHODS: To test this hypothesis, isolated Langendorff-perfused rabbit hearts were subjected to 45 min global ischemia followed by 120 min reperfusion with IPC (n=0) or without IPC (ischemic control, n=). IPC was induced by one cycle of 5 min global ischemia and 10 min reperfusion. In the glucose (G)-free preconditioned group (n=0), G depletion-repletion was induced by perfusion with G-free Tyrode solution for 5 min and then G-containing Tyrode solution for 10 min followed by 45 min ischemia and 120 min reperfusion. For glycogen depletion or loading, hearts were treated with sodium acetate (NA, 5 mM, n=) or insulin (Ins, 1 unit/L, n=) for 15 min before 45 min ischemia. Left ventricular function and coronary flow (CF) were continuously recorded during experiments. Myocardial cytosolic and membrane protein kinase C (PKC) activities were measured by 32P-gamma-ATP incorporation into PKC-specific pepetide; glycogen content in the cardiac myocytes was determined by spectrophotometry with amyloglucosidase; expression of PKC isozymes was determined by Western blot with monoclonal antibodies. Infarct size was determined by staining with tetrazolium salt and planimetry. Data were analyzed by ANOVA and Tukey's post-hoc test. RESULTS: IPC or G-free preconditioning enhanced LV functional recovery; NA did not influence on functional recovery but Ins depressed it. Infarct size was significantly reduced by IPC, G-free preconditioning, and NA treatment (35.3+/-2.1% in the ischemic control, 18.7+/-1.2% in the IPC, 22.1+/-1.2% in the G-free preconditioned, 16.3+/-1.2% in the NA-treated group, and 32.8+/-1.6% in the Ins-treated group, p<0.05). Membrane PKC activities significantly increased by IPC, IPC and 45 min ischemia, G-free preconditioning, and G-free preconditioning and 45 min ischemia; especially, expression of membrane PKC-epsilon increased by IPC and G-free preconditioning. Glycogen content decreased by 45 min ischemia, IPC, G-free preconditioning, and by NA treatment, but increased by Ins treatment. CONCLUSION: These results suggest that in rabbit heart, intracellular glycogen may not significantly be related with the cardioprotective effect of IPC; G-free preconditioning could not improve post-ischemic contractile dysfunction but it has an infarct size-limiting effect; this cardioprotective effect may be related in part to activation of PKC, especially epsilon isozyme.
Animals ; Antibodies, Monoclonal ; Blotting, Western ; Cytosol ; Glucan 1,4-alpha-Glucosidase ; Glucose ; Glycogen* ; Heart ; Insulin ; Ischemia ; Ischemic Preconditioning* ; Isoenzymes ; Membrane Proteins ; Membranes ; Myocytes, Cardiac ; Perfusion ; Phosphotransferases ; Protein Kinase C* ; Protein Kinases* ; Rats ; Reperfusion ; Sodium Acetate ; Spectrophotometry ; Ventricular Function, Left

It is clear that creatine plays a reservoir of high energy phosphate bond as creatine phosphate and maintains ATP levels in skeletal muscle and nervous tissues. Creatine and creatine kinase activity are required to utilize creatine phosphate as high energy phosphate. The contents of creatine in testis of rats were determined by the method of Van Pilsum and compared with other organs for the study of the physiological role of creatine in testis. Creatine content of tests was 39.82+/-3.36 ug/g wet tissue compared with skeletal muscle, 52.92+/-10.25 ug/g wet tissue. It was relatively high compared with brain (15.45+/-6.49 ug/g wet tissue), heart (20.0+/-2.91 ug/g wet tissue), kidney (29.55+/-2.52 ug/g wet tissue) and liver (12.68+/-1.94 ug/g wet tissue). Creatinine content of testes (45.84+/-4.08 ug/g wet tissue) was very high, compared with skeletal muscle (24.14+/-7.73 ug/g wet tissue), heart .(23.71+/-4.73 ug/g wet tissue), brain (17.24+/-1.19 ug/g wet tissue), kidney (14.92+/-3.45 ug/g set tissue), and liver (9.59 +/-1.26 ug/g wet tissue). I suppose that creatine in testis of rats may be a part of potent system for generation of ATP from ADP hydrolyzing creatine phosphate.
Adenosine Diphosphate ; Adenosine Triphosphate ; Animals ; Brain ; Creatine Kinase ; Creatine* ; Creatinine ; Heart ; Kidney ; Liver ; Muscle, Skeletal ; Phosphocreatine ; Rats* ; Testis

The atrial acetylcholine-activated K+ (KACh) channel is gated by the pertussis toxin-sensitive inhibitory G (GK) protein. Earlier studies revealed that ATP alone can activate the KACh channel via transphosphorylation mediated by nucleoside-diphosphate kinase (NDPK) in atrial cells of rabbit and guinea pig. This channel can be activated by various agonists and also modulated its function by phosphorylation. ATP-induced KACh channel activation (AIKA) was maintained in the presence of the NDPK inhibitor, suggesting the existence of a mechanism other than NDPK-mediated process. Here we hypothesized the phosphorylation process as another mechanism underlying AIKA and was undertaken to examine what kinase is involved in atrial cells isolated from the rat heart. Single application of 1 mM ATP gradually increased the activity of KACh channels and reached its maximum 40 ~ 50 sec later following adding ATP. AIKA was not completely reduced but maintained by half even in the presence of NDPK inhibitor. Neither ADP nor a non-hydrolyzable ATP analogue, AMP-PNP can cause AIKA, while a non-specific phosphatase, alkaline phosphatase blocked completely AIKA. PKC antagonists such as sphingosine or tamoxifen, completely blocked AIKA, whereas PKC catalytic domain increased AIKA. Taken together, it is suggested that the PKC-mediated phosphorylation is partly involved in AIKA.
Adenosine Diphosphate ; Adenosine Triphosphate ; Adenylyl Imidodiphosphate ; Alkaline Phosphatase ; Animals ; Catalytic Domain ; Guinea Pigs ; Heart ; Nucleoside-Diphosphate Kinase ; Phosphorylation ; Phosphotransferases ; Protein Kinase C* ; Protein Kinases* ; Rats ; Sphingosine ; Tamoxifen ; Whooping Cough

Adenosine is a naturally occurring breakdown product of adenosine triphosphate and plays an important role in different physiological and pathological conditions. Adenosine also serves as an important trigger in ischemic and remote preconditioning and its release may impart cardioprotection. Exogenous administration of adenosine in the form of adenosine preconditioning may also protect heart from ischemia-reperfusion injury. Endogenous release of adenosine during ischemic/remote preconditioning or exogenous adenosine during pharmacological preconditioning activates adenosine receptors to activate plethora of mechanisms, which either independently or in association with one another may confer cardioprotection during ischemia-reperfusion injury. These mechanisms include activation of K(ATP) channels, an increase in the levels of antioxidant enzymes, functional interaction with opioid receptors; increase in nitric oxide production; decrease in inflammation; activation of transient receptor potential vanilloid (TRPV) channels; activation of kinases such as protein kinase B (Akt), protein kinase C, tyrosine kinase, mitogen activated protein (MAP) kinases such as ERK 1/2, p38 MAP kinases and MAP kinase kinase (MEK 1) MMP. The present review discusses the role and mechanisms involved in adenosine preconditioning-induced cardioprotection.
Adenosine Triphosphate ; Adenosine* ; Heart ; Inflammation ; Mitogen-Activated Protein Kinase Kinases ; Nitric Oxide ; Phosphotransferases ; Protein Kinase C ; Protein-Tyrosine Kinases ; Proto-Oncogene Proteins c-akt ; Receptors, Opioid ; Receptors, Purinergic P1 ; Reperfusion Injury

BACKGROUND AND OBJECTIVES: The myocardial protective effect of ischemic preconditioning is well known. However, the mechanism is remains unclear. The purpose of this study is to determine the role of adenosine, protein kinase C, KATP channel and the change of monophasic action potential duration on cardioprotective effect of ischemic preconditioning in cat. Materials AND METHODS: In this experiment, 66 cats were allocated into 7 groups:control (n=10), ischemic preconditioning (n=10), adenosine pre-treated (n=10), SPT (8-p-sulfophenyl theophylline) pre-treated (n=9), polymyxin B pre-treated (n=9), glibenclamide pre-treated (n=9) and nicorandil pre-treated (n=9) groups. Ischemic preconditioning was performed in ischemic preconditioning, SPT pre-treated, polymyxin B pre-treated and glibenclamide pre-treated groups by 3 episodes of 5 minutes ischemia and 10 minutes reperfusion. All animals were subjected to 40 minutes of ischemia and 40 minutes reperfusion. Monophasic action potential duration at 50% repolarization (MAP50) was measured in the ischemic and non-ischemic area respectively by epicardial probe throughout the experiment. The effect of ischemic preconditioning was determined by infarct size (% area at risk). RESULTS: Ischemic preconditioning, adenosine pre-treatment and nicorandil pre-treatment groups demonstrated a significant reduction in infarct size (26+/-4%, 25+/-4% and 34+/-8% infarction of the risk zone, respectively, p<0.01, p<0.01 and p<0.05 vs. control) with respect to control (41+/-8% infarction of the risk zone). However, pretreatment with SPT, polymyxin B or glibenclamide abolished the effect of ischemic preconditioning. Ischemic preconditioning group exhibited a significant reduction of MAP50 duration in the ischemic area during preconditioning;at the first preconditioning 128+/-11 msec vs. 144+/-10 msec control, at the second preconditioning 110+/-10 msec vs.147+/-10 msec control (p<0.01), at the third preconditioning 114+/-10 msec vs. 145+/-11 msec control (p<0.05). But, pretreatment with SPT, polymyxin B and glibenclamide prevented the reduction of MAP50 in the ischemic area during ischemic preconditioning. During 40 minutes ischemia, the shortening of MAP50 was more pronounced in the preconditioned group than in control group;at 5 minutes 112+/-13 msec vs. 124+/-10 msec control, at 10 minutes 89+/-12 msec vs. 133+/-11 msec control (p<0.05 ), at 20 minutes 93+/-12 msec vs. 136+/-11 msec control (p<0.05), and at 30 minutes 107+/-19 msec vs. 144+/-14 msec control (p<0.05). In adenosine pre-treated group, the MAP50 was significantly shortened than control group throughout 40 minutes occlusion period;at 5 minutes 90+/-8 msec (p<0.05), at 10 minutes 77+/-9 msec (p<0.05), at 20 minutes 92+/-8 msec (p<0.05), and at 30 minutes 103+/-8 msec (p<0.05). Nicorandil pretreatment pronounced the ischemic shortening of MAP50 in ischemic area and the effect was significant during early ischemic period;at 10 minutes 98+/-22 msec (p<0.05 vs. control). In pretreatment groups with SPT, polymyxin B or glibenclamide, the ischemic preconditioning of MAP50 measured in non-ischemic area was not significantly different compared with control group. MAP50 measured in ischemic area during reperfusion was not significantly different between groups. CONCLUSION: Based on this study, adenosine receptor-protein kinase C-KATP channel activation and monophasic action potential duration shortening during ischemia play an important role in myocardial protection during ischemic injury.
Action Potentials* ; Adenosine* ; Animals ; Cats* ; Glyburide ; Infarction ; Ischemia ; Ischemic Preconditioning* ; Nicorandil ; Phosphotransferases ; Polymyxin B ; Protein Kinase C* ; Protein Kinases* ; Receptors, Purinergic P1* ; Reperfusion

PURPOSE: To investigate the signal enhancement ratio by NOE effect on in vivo 31P MRS in human heart muscle and liver. we also evaluated the enhancement ratios of different phosphorus metabolites, which are important in 31P MRS for each organ. MATERIALS AND METHODS: Ten normal subjects (M: F = 8: 2, age range = 24-32 yrs) were included for in vivo 31P MRS measurements on a 1.5 T whole-body MRI/MRS system using 1H-31P dual tuned surface coil. Two-dimensional Chemical Shift Imaging (2D CSI) pulse sequence for 31P MRS was employed in all 31P MRS measurements. First, 31P MRS performed without NOE effect and then the same 2D CSI data acquisitions were repeated with NOE effect. After postprocessing the MRS raw data in the time domain, the signal enhancements in percent were estimated from the major metabolites. RESULTS: The calculated NOE enhancement for liver 31P MRS were: alpha-ATP (7%), beta- ATP (9%), gamma-ATP (17%), Pi (1%), PDE (19%), and PME (31%). Because there is no creatine kinase activity in liver, PCr signal is absent. For cardiac 31P MRS, whole body coil gave better scout images and thus better localization than surface coil. In 31P cardiac multi-voxel spectra, DPG signal increased from left to right according to the amount of blood included. The calculated enhancement for cardiac 31P MRS were: alpha -ATP (12%), beta-ATP (19%), gamma-ATP (30%), PCr (34%), Pi (20%), PDE (51%), and DPG (72%). CONCLUSION: Our results revealed that the NOE effect was more pronounced in heart muscle than in liver with different coupling to 1H spin system and thus different heteronuclear cross-relaxation.
Adenosine Triphosphate ; Creatine Kinase ; Heart* ; Humans ; Liver* ; Magnetic Resonance Imaging ; Myocardium ; Phosphorus ; Polymerase Chain Reaction

OBJECTIVETo investigate the effect of sphingosine kinase 1 (SphK1) on the proliferation, apoptosis, migration and invasion of colon cancer TH-29 cells and to explore its molecular mechanisms.

METHODSPhorbol 12-myristate 13-acetate (PMA) was used to induce the activity of SphK1 and N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. Cell prolieration and apoptosis were detected by MTT assay and flow cytometry, respectively. The migration and invasion capabilities of the cells were assessed in Transwell chambers. The activity of SphK1 was assayed by autoradiography. Western blot was used to evaluate the protein expression of SphK1, p38, phosphorylated p38 (p-p38) and SAPK/JNK.

RESULTSPMA and DMS were able to induce and suppress the activity and protein expression of SphK1 in a time-dependent manner, respectively. PMA enhanced and DMS suppressed the cell viability in a time- and dose-dependent manner. Being treated with 100 nmol/L PMA or 50 µmol/L DMS for 0, 6, 12, 24 h, the cell apoptosis rates of PMA group were (9.35 ± 0.84)%, (7.61 ± 0.48)%, (5.53 ± 0.76)% and (0.56 ± 0.33)%, contrastly, that of DMS group were (9.18 ± 0.94)%, (12.06 ± 1.41)%, (19.80 ± 2.36)% and (31.85 ± 3.60)%, respectively. Compared with the control group, the cell migration and invasion capabilities of the PMA group were significantly enhanced, and that of the DMS group were significantly suppressed. The migration cell number of control, PMA and DMS groups were 68.75 ± 6.15, 109.33 ± 11.63 and 10.83 ± 2.48, the invasion cell number of control, PMA and DMS groups were 55.42 ± 4.50, 90.58 ± 7.06 and 9.58 ± 2.39, respectively. With the elevating activity and expression of SphK1, the protein expressions of p38, p-p38 and SAPK/JNK were strikingly suppressed. On the contrary, after treating with DMS the protein expressions of p38, p-p38 and SAPK/JNK were enhanced.

CONCLUSIONSSphK1 potently enhances the prolieration, migration and invasion of colon cancer HT-29 cells, meanwhile suppresses the cell apoptosis. The suppressing of the p38 and SAPK/JNK signalling pathways may be one of its molecular mechanisms.

Apoptosis ; drug effects ; Carcinogens ; administration & dosage ; pharmacology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; administration & dosage ; pharmacology ; HT29 Cells ; Humans ; MAP Kinase Kinase 4 ; metabolism ; Neoplasm Invasiveness ; Phosphorylation ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; physiology ; Sphingosine ; administration & dosage ; analogs & derivatives ; pharmacology ; Tetradecanoylphorbol Acetate ; administration & dosage ; analogs & derivatives ; pharmacology ; Time Factors ; p38 Mitogen-Activated Protein Kinases ; metabolism