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1.
PLoS One ; 12(3): e0172965, 2017.
Article in English | MEDLINE | ID: mdl-28257486

ABSTRACT

PURPOSE: Exertional rhabdomyolysis can occur in individuals performing various types of exercise but it is unclear why some individuals develop this condition while others do not. Previous investigations have determined the role of several single nucleotide polymorphisms (SNPs) to explain inter-individual variability of serum creatine kinase (CK) concentrations after exertional muscle damage. However, there has been no research about the interrelationship among these SNPs. The purpose of this investigation was to analyze seven SNPs that are candidates for explaining individual variations of CK response after a marathon competition (ACE = 287bp Ins/Del, ACTN3 = p.R577X, CKMM = NcoI, IGF2 = C13790G, IL6 = 174G>C, MLCK = C37885A, TNFα = 308G>A). METHODS: Using Williams and Folland's model, we determined the total genotype score from the accumulated combination of these seven SNPs for marathoners with a low CK response (n = 36; serum CK <400 U·L-1) vs. marathoners with a high CK response (n = 31; serum CK ≥400 U·L-1). RESULTS: At the end of the race, low CK responders had lower serum CK (290±65 vs. 733±405 U·L-1; P<0.01) and myoglobin concentrations (443±328 vs. 1009±971 ng·mL-1, P<0.01) than high CK responders. Although the groups were similar in age, anthropometric characteristics, running experience and training habits, total genotype score was higher in low CK responders than in high CK responders (5.2±1.4 vs. 4.4±1.7 point, P = 0.02). CONCLUSION: Marathoners with a lower CK response after the race had a more favorable polygenic profile than runners with high serum CK concentrations. This might suggest a significant role of genetic polymorphisms in the levels of exertional muscle damage and rhabdomyolysis. Yet other SNPs, in addition to exercise training, might also play a role in the values of CK after damaging exercise.


Subject(s)
Creatine Kinase, MM Form/genetics , Physical Exertion , Polymorphism, Single Nucleotide , Rhabdomyolysis/diagnosis , Rhabdomyolysis/genetics , Actinin/blood , Actinin/genetics , Adolescent , Adult , Aged , Creatine Kinase, MM Form/blood , Female , Gene Expression Profiling , Gene Expression Regulation , Humans , Insulin-Like Growth Factor II/genetics , Insulin-Like Growth Factor II/metabolism , Interleukin-6/blood , Interleukin-6/genetics , Male , Middle Aged , Myoglobin/blood , Myosin-Light-Chain Kinase/blood , Myosin-Light-Chain Kinase/genetics , Peptidyl-Dipeptidase A/blood , Peptidyl-Dipeptidase A/genetics , Prognosis , Rhabdomyolysis/blood , Rhabdomyolysis/pathology , Running , Tumor Necrosis Factor-alpha/blood , Tumor Necrosis Factor-alpha/genetics
2.
Circ J ; 80(1): 218-26, 2016.
Article in English | MEDLINE | ID: mdl-26549004

ABSTRACT

BACKGROUND: To better understand the evolution of typical hypertrophic cardiomyopathy (HCM) to heart failure (HF), we investigated the relationship between serum biochemical abnormalities and changes in left ventricular (LV) remodeling. METHODS AND RESULTS: Seventy-seven HCM patients were followed for 20 years. Creatine kinase (CK), CK-MB, lactate dehydrogenase (LDH), LDH-1, troponin T and myosin light chain-1 (MLC-1) were measured. Abnormal CK-MB elevation was observed in 64% of HCM patients. LDH-1 was not significantly different compared with the control subjects. Troponin T elevation was observed in 3 HCM patients and MLC-1 elevation was not observed. According to median CK-MB, HCM patients were divided into 2 groups: group H (CK-MB ≥2.5%, n=33) and group L (CK-MB <2.5%, n=44). During the follow-up period in group H, LV end-diastolic dimension increased (P<0.0001), fractional shortening decreased (P<0.0004), and left atrial dimension increased (P<0.0001). The markers reflecting LV hypertrophy were significantly decreased. In group L, LV end-diastolic dimension increased (P<0.02) and left atrial dimension increased (P<0.0001). HF was observed in 18 patients in group H and in 4 in group L. There were 14 HF deaths in group H and 2 in group L, and 3 sudden cardiac deaths in group H. CONCLUSIONS: Persistent elevation of cardiac enzymes in HCM patients indicates ongoing myocardial injury, ultimately resulting in death by HF.


Subject(s)
Cardiomegaly , Heart Failure , L-Lactate Dehydrogenase/blood , Muscle Proteins/blood , Myosin-Light-Chain Kinase/blood , Troponin T/blood , Adult , Aged , Aged, 80 and over , Cardiomegaly/blood , Cardiomegaly/pathology , Female , Follow-Up Studies , Heart Failure/blood , Heart Failure/pathology , Humans , Isoenzymes/blood , Male , Middle Aged , Prospective Studies
3.
Ann Clin Lab Sci ; 45(1): 54-7, 2015.
Article in English | MEDLINE | ID: mdl-25696011

ABSTRACT

The present study was designed to identify changes in human serum caused by myosin light chain kinase (MLCK) in the context of Type 2 diabetes mellitus (T2DM), and to determine whether it had correlations with other biomarkers of T2DM. A total of 336 patients with T2DM and 90 sex- and age-matched, apparently healthy subjects were selected. The serum MLCK of all participants was detected by quantitative ELISA. Our results showed that the concentration of MLCK in the T2DM group was significantly higher than in control samples, and there were no correlations with age, FBG, TC, HDL-C, LDL-C or HbA1c. These findings suggest that serum MLCK is associated with T2DM, which gives some clues for identifying new biomarkers for the diagnosis of T2DM and its complications.


Subject(s)
Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/enzymology , Myosin-Light-Chain Kinase/blood , Case-Control Studies , Cross-Sectional Studies , Female , Humans , Male , Middle Aged
4.
Blood ; 122(20): 3533-45, 2013 Nov 14.
Article in English | MEDLINE | ID: mdl-24100445

ABSTRACT

Cyclic adenosine monophosphate (cAMP)-dependent signaling modulates platelet shape change through unknown mechanisms. We examined the effects of cAMP signaling on platelet contractile machinery. Prostaglandin E1 (PGE1)-mediated inhibition of thrombin-stimulated shape change was accompanied by diminished phosphorylation of myosin light chain (MLC). Since thrombin stimulates phospho-MLC through RhoA/Rho-associated, coiled-coil containing protein kinase (ROCK)-dependent inhibition of MLC phosphatase (MLCP), we examined the effects of cAMP on this pathway. Thrombin stimulated the membrane localization of RhoA and the formation of a signaling complex of RhoA/ROCK2/myosin phosphatase-targeting subunit 1 (MYPT1). This resulted in ROCK-mediated phosphorylation of MYPT1 on threonine 853 (thr(853)), the disassociation of the catalytic subunit protein phosphatase 1δ (PP1δ) from MYPT1 and inhibition of basal MLCP activity. Treatment of platelets with PGE1 prevented thrombin-induced phospho-MYPT1-thr(853) in a protein kinase A (PKA)-dependent manner. Examination of the molecular mechanisms revealed that PGE1 induced the phosphorylation of RhoA on serine(188) through a pathway requiring cAMP and PKA. This event inhibited the membrane relocalization of RhoA, prevented the association of RhoA with ROCK2 and MYPT1, attenuated the dissociation of PP1δ from MYPT1, and thereby restored basal MLCP activity leading to a decrease in phospho-MLC. These data reveal a new mechanism by which the cAMP-PKA signaling pathway regulates platelet function.


Subject(s)
Blood Platelets/enzymology , Cyclic AMP/physiology , Myosin-Light-Chain Kinase/metabolism , Myosin-Light-Chain Phosphatase/physiology , Protein Processing, Post-Translational/physiology , Second Messenger Systems/physiology , Signal Transduction/physiology , rho-Associated Kinases/physiology , rhoA GTP-Binding Protein/physiology , Alprostadil/pharmacology , Blood Platelets/drug effects , Blood Platelets/ultrastructure , Cell Shape/drug effects , Cell Shape/physiology , Cyclic AMP-Dependent Protein Kinases/physiology , Humans , In Vitro Techniques , Multiprotein Complexes , Myosin-Light-Chain Kinase/blood , Phosphorylation , Phosphoserine/metabolism , Phosphothreonine/metabolism , Protein Phosphatase 1/metabolism , Protein Subunits , Thrombin/pharmacology
5.
Dis Markers ; 32(3): 195-202, 2012.
Article in English | MEDLINE | ID: mdl-22377736

ABSTRACT

Most cases of gastric cancer (GC) are not diagnosed at early stage which can be curable, so it is necessary to identify effective biomarkers for its diagnosis and pre-warning. We have used methylated DNA immunoprecipitation (MeDIP) to identify genes that are frequently methylated in gastric cancer cell lines. Promoter regions hypermethylation of candidate genes were tested by methylation-specific polymerase chain reaction (MSP) in serum samples, including GC (n=58), gastric precancerous lesions (GPL, n=46), and normal controls (NC, n=30). Eighty two hypermethylated genes were acquired by array analysis and 5 genes (BCAS4, CHRM2, FAM5C, PRAC and MYLK) were selected as the candidate genes. Three genes (CHRM2, FAM5C and MYLK) were further confirmed to show methylation rates increased with progression from NC to GPL, then to GC. There was obvious decrease in detection of FAM5C and MYLK hypermethylation, but not CHRM2, from preoperative to postoperative evaluation (P< 0.001). Combined detection of FAM5C and MYLK hypermethylation had a higher sensitivity in GC diagnosis (77.6%,45/58) and pre-warning (30.4%,14/46) than one single gene detection and also had a high specificity of 90%. The combined hypermethylated status of FAM5C and MYLK correlated with tumor size (P<0.001), tumor invasion depth (P=0.001) and tumor-node-metastasis (TNM) stage (P=0.003). Hypermethylated FAM5C and MYLK can be used as potential biomarkers for diagnosis and pre-warning of GC.


Subject(s)
Calcium-Binding Proteins/genetics , DNA-Binding Proteins/genetics , Myosin-Light-Chain Kinase/genetics , Precancerous Conditions , Stomach Neoplasms/diagnosis , Stomach/pathology , Calcium-Binding Proteins/blood , Case-Control Studies , Cell Line, Tumor , DNA Methylation , DNA-Binding Proteins/blood , Early Diagnosis , Gastric Mucosa/metabolism , Humans , Middle Aged , Myosin-Light-Chain Kinase/blood , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Precancerous Conditions/blood , Precancerous Conditions/diagnosis , Precancerous Conditions/pathology , Promoter Regions, Genetic , Sensitivity and Specificity , Stomach Neoplasms/blood , Stomach Neoplasms/pathology , Tumor Burden
6.
BMC Gastroenterol ; 10: 68, 2010 Jun 29.
Article in English | MEDLINE | ID: mdl-20587030

ABSTRACT

BACKGROUND: Pancreatic cancer has significant morbidity and mortality worldwide. Good prognosis relies on an early diagnosis. The purpose of this study was to develop techniques for identifying cancer biomarkers in the serum of patients with pancreatic cancer. METHODS: Serum samples from five individuals with pancreatic cancer and five individuals without cancer were compared. Highly abundant serum proteins were depleted by immuno-affinity column. Differential protein analysis was performed using 2-dimensional differential in-gel electrophoresis (2D-DIGE). RESULTS: Among these protein spots, we found that 16 protein spots were differently expressed between the two mixtures; 8 of these were up-regulated and 8 were down-regulated in cancer. Mass spectrometry and database searching allowed the identification of the proteins corresponding to the gel spots. Up-regulation of mannose-binding lectin 2 and myosin light chain kinase 2, which have not previously been implicated in pancreatic cancer, were observed. In an independent series of serum samples from 16 patients with pancreatic cancer and 16 non-cancer-bearing controls, increased levels of mannose-binding lectin 2 and myosin light chain kinase 2 were confirmed by western blot. CONCLUSIONS: These results suggest that affinity column enrichment and DIGE can be used to identify proteins differentially expressed in serum from pancreatic cancer patients. These two proteins 'mannose-binding lectin 2 and myosin light chain kinase 2' might be potential biomarkers for the diagnosis of the pancreatic cancer.


Subject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Mannose-Binding Lectin/blood , Myosin-Light-Chain Kinase/blood , Pancreatic Neoplasms/blood , Pancreatic Neoplasms/diagnosis , Proteomics , Up-Regulation/physiology , Adenocarcinoma/blood , Adenocarcinoma/diagnosis , Adult , Aged , Biomarkers, Tumor/blood , CA-19-9 Antigen/blood , Carcinoembryonic Antigen/blood , Case-Control Studies , Female , Humans , Male , Middle Aged , Tandem Mass Spectrometry
7.
J Thromb Haemost ; 8(3): 588-95, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20002541

ABSTRACT

BACKGROUND: We have used the advantages of the zebrafish model system to demonstrate which of the vertebrate myosin light chain kinase (MLCK) genes is expressed in thrombocytes and important for thrombus formation. METHODS AND RESULTS: Here we report that Mlck1a is an essential component of thrombus formation. Phylogenetic data revealed four zebrafish orthologous for three human MLCK genes. To investigate expression of the zebrafish mlck genes in thrombocytes we compared GFP-tagged platelets with other cells by microarray analysis, and showed that mlck1a expression was 4.5-fold enriched in platelets. Furthermore, mlck1a mRNA and mRNA for the platelet-specific cd41 co-localized in thrombi. Expression of other mlck subtypes was lower in GFP-tagged platelets (mlck1b; 0.77-fold enriched) and absent in thrombi (mlck1b, -2, -3). To investigate the role of Mlck1a in thrombus formation, we knocked down mlck1a using two morpholinos. This resulted in impaired morphology changes of platelets adhering on fibrinogen. In a thrombosis model, in which thrombocytes adhere to the vessel wall damaged by laser irradiation, thrombus formation was slowed down in mlck1a-deficient embryos. CONCLUSION: We conclude that Mlck1a is the subtype of MLCK that contributes to platelet shape change and thrombus formation.


Subject(s)
Blood Platelets/enzymology , Myosin-Light-Chain Kinase/blood , Thrombosis/enzymology , Zebrafish Proteins/blood , Zebrafish/blood , Animals , Animals, Genetically Modified , Cell Shape , Disease Models, Animal , Fibrinogen/metabolism , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Gene Knockdown Techniques , Myosin-Light-Chain Kinase/genetics , Oligonucleotide Array Sequence Analysis , Platelet Adhesiveness , Platelet Membrane Glycoprotein IIb/blood , RNA, Messenger/blood , Recombinant Fusion Proteins/blood , Thrombosis/blood , Thrombosis/genetics , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/genetics
8.
J Leukoc Biol ; 83(4): 964-71, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18218860

ABSTRACT

Activation of beta2 integrins is necessary for neutrophil adhesion and full activation of neutrophil effector functions. We demonstrated previously that inhibition of protein kinase A (PKA) activity in quiescent neutrophils is sufficient to increase beta2-integrin cell surface expression, affinity, and adhesion. Thus, a tonic level of PKA activity prevents inappropriate activation of beta2 integrins in unstimulated neutrophils. Myosin light-chain (MLC) phosphorylation is an important regulator of leukocyte integrin function and adhesion. Moreover, PKA regulates MLC phosphorylation via inhibiting MLC kinase (MLCK) and MLC dephosphorylation via effects on the Rho kinase (ROCK)/MLC phosphatase pathway. We hypothesize that the tonic inhibitory effect of PKA on beta2-integrin activation neutrophils operates via its inhibition of MLC phosphorylation. We demonstrate here that inhibition of PKA activity with KT5720 activated beta2 integrins and adhesion coincident with an increase in MLC serine 19 (Ser 19) phosphorylation. KT5720-induced activation of beta2 integrins, adhesion, and MLC Ser 19 phosphorylation was abolished by pretreatment with the MLCK inhibitor ML-7 and specific MLCK inhibitory peptides but not the ROCK inhibitor Y-27632. These findings demonstrate that tonic PKA activity prevents activation of beta2 integrins and adhesion by inhibiting MLC phosphorylation via a MLCK-dependent but ROCK-independent pathway.


Subject(s)
CD18 Antigens/blood , Cyclic AMP-Dependent Protein Kinases/blood , Myosin-Light-Chain Kinase/blood , Neutrophils/physiology , rho-Associated Kinases/blood , Adult , CD18 Antigens/drug effects , Carbazoles/pharmacology , Cell Adhesion , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Homeostasis , Humans , Indoles/pharmacology , Neutrophils/enzymology , Oligopeptides/pharmacology , Phosphorylation , Pyrroles/pharmacology
9.
Article in English | MEDLINE | ID: mdl-14687859

ABSTRACT

Serotonin (5-HT)- or thrombin-stimulated platelet intracellular calcium (Ca) mobilization has been reported to be enhanced in patients with bipolar disorders. However, the mechanism of this enhancement is unknown. As a preliminary study, the authors examined the effects of a myosin light chain kinase (MLCK) inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9), and two drugs that are mainstays of treatment for bipolar disorder, lithium and valproate, on 5-HT- or thrombin-induced Ca increase in the platelets of normal subjects. When preincubated with 30 microM ML-9, Ca responses to both agonists were enhanced. Valproate showed a dose-dependent attenuation of agonist-induced intracellular Ca rise, both in the absence and presence of ML-9. Although lithium alone had no significant effect on the Ca increase, a high concentration of lithium significantly decreased Ca mobilization only in the presence of ML-9. These results suggest that the enhanced Ca response observed in bipolar disorder might be relevant to decreased function of MLCK and that the mechanism of action of lithium may include a compensatory effect on MLCK modulation.


Subject(s)
Antimanic Agents/pharmacology , Blood Platelets/metabolism , Calcium/blood , Lithium/pharmacology , Myosin-Light-Chain Kinase/blood , Valproic Acid/pharmacology , Adult , Azepines/pharmacology , Blood Platelets/drug effects , Enzyme Inhibitors/pharmacology , Humans , In Vitro Techniques , Male , Myosin-Light-Chain Kinase/antagonists & inhibitors , Pilot Projects , Serotonin/pharmacology , Thrombin/pharmacology
10.
J Leukoc Biol ; 54(4): 351-9, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8409758

ABSTRACT

Monocyte chemotaxis is severely depressed in patients with advanced tumors, but the cellular basis for this chemotactic defect is not known. Because the actomyosin cytoskeleton is thought to play a primary role in chemotaxis, we have employed flow cytometry to examine several aspects of the contractile machinery including myosin II, myosin light chain kinase (MLCK), actin, and cytoplasmic calcium in unstimulated and in formylpeptide-stimulated neutrophils and monocytes. Serum-pretreated polymorphonuclear leukocytes (PMNs) and monocytes from healthy blood donors or PMNs and monocytes isolated from tumor patients were studied. Leukocytes pretreated with serum from cancer patients exhibited decreased baseline myosin staining and a vastly different response to formylpeptide stimulation compared with leukocytes pretreated with normal human serum. In contrast, similar amounts of MLCK were observed in neutrophils and monocytes preincubated with normal or cancer serum with or without stimulation with formylpeptide. The fluorescent calcium indicator fluo-3 showed that resting and fMLP-stimulated levels of intracellular calcium were not significantly different in control and cancer serum-pretreated human leukocytes or in leukocytes isolated from tumor patients. Similarly, resting and fMLP-stimulated levels of F-actin in cancer patients' leukocytes as assessed by NBD-phallacidin staining did not differ significantly from those of normal leukocytes. Because the actomyosin cytoskeleton is intricately involved in leukocyte chemotaxis, alterations in the cytoskeleton may dramatically affect cell motility. The cytoskeletal alterations and changes in the response of leukocytes pretreated with cancer patients' serum to formylpeptide stimulation as described here may result in decreased chemotaxis by these cells.


Subject(s)
Calcium/blood , Chemotaxis, Leukocyte , Cytoskeleton/ultrastructure , Head and Neck Neoplasms/blood , Monocytes/physiology , Monocytes/ultrastructure , Neutrophils/physiology , Neutrophils/ultrastructure , Actins/blood , Adult , Chemotaxis, Leukocyte/drug effects , Humans , In Vitro Techniques , Kinetics , Monocytes/drug effects , Myosin-Light-Chain Kinase/blood , Myosins/blood , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Reference Values
11.
J Cell Physiol ; 155(3): 505-19, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8388002

ABSTRACT

Activation of neutrophils results in morphological and functional alterations including changes in cell shape and initiation of motile behavior that depend on assembly and reorganization of the actin cytoskeleton. Phosphoproteins are thought to be key intermediates in the regulation of cytoskeletal alterations and whereas much attention has been directed at the role of protein kinases, relatively little information is available on the importance of phosphatases. To elucidate the role of protein phosphatases, we studied the effects of the phosphatase inhibitors okadaic acid and calyculin A on the actin cytoskeleton of human neutrophils. Exposure of cells to okadaic acid resulted in assembly and spatial redistribution of actin, which peaked at 25 min and returned to baseline levels by 45 min, as assessed by flow cytometric analysis of NBD-phallacidin stained cells and confocal fluorescence microscopy, respectively. These effects correlated with an increase in protein phosphorylation, determined by incorporation of 32P into cellular proteins using SDS-PAGE and autoradiography. Similar but more rapid responses were observed in electropermeabilized cells treated with okadaic acid or calyculin A. The dose dependence of these effects was compatible with a role for phosphatase type 1 as the target enzyme. These findings also suggested the presence of constitutively active protein kinases capable of effecting actin polymerization. Phosphorylation of myosin light chain (MLC) has been postulated to promote actin assembly, but myosin light chain kinase (MLCK) appeared not to be involved because: (1) the effect of okadaic acid was not inhibited by the MLCK inhibitor KT5926 and (2) in permeabilized cells suspended in medium with free calcium [Ca2+] < 10 nM (conditions under which MLCK is inactive), the effect of okadaic acid persisted. The role of phosphatases in stimulus-induced actin assembly was assessed in cells preincubated with okadaic acid for 45 min, after F-actin levels had returned to baseline. Under these conditions, okadaic acid completely abrogated actin assembly induced by phorbol myristate acetate, platelet activating factor, and leukotriene B4, whereas the effects of the chemotactic peptide fMLP and opsonized zymosan (OpZ) were unaffected. We conclude that serine and threonine phosphatases exert a tonic negative influence on actin assembly and organization. Furthermore, divergent pathways seem to mediate the response to lipidic stimuli, on one hand, and fMLP and OpZ, on the other, as evidenced by the differential susceptibility to inhibition by okadaic acid.


Subject(s)
Actins/blood , Ethers, Cyclic/pharmacology , Neutrophils/metabolism , Phosphoprotein Phosphatases/blood , Adenosine Triphosphate/pharmacology , Cell Movement , Cytoskeleton/drug effects , Cytoskeleton/metabolism , GTP-Binding Proteins/physiology , Humans , Marine Toxins , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Molecular Weight , Myosin-Light-Chain Kinase/blood , Neutrophils/drug effects , Neutrophils/ultrastructure , Okadaic Acid , Oxazoles/pharmacology , Phosphoproteins/metabolism , Protein Kinase C/blood , Protein Kinases/blood , Receptors, Cell Surface/physiology
13.
Thromb Res ; 59(3): 475-87, 1990 Aug 01.
Article in English | MEDLINE | ID: mdl-2173166

ABSTRACT

This study investigates the role of protein kinase C and of myosin light chain kinase in mediating platelet hyperresponsiveness in spontaneously hypertensive rats (SHR). For this purpose, 32P-labeled washed platelets of both SHR and normotensive controls Wistar-Kyoto (WKY) were challenged either with a receptor-mediated agonist (thrombin) or with direct activators of myosin light chain kinase and protein kinase C. Such enzymatic activities were assessed by measuring changes in 32P-labeling of their respective target proteins, namely myosin light chain (20 KDa) and the 47 KDa protein. In resting platelets, the patterns of protein phosphorylation were similar between SHR and WKY, suggesting that the two cell types were in a comparable quiescent status. By contrast, in both dose-response and time-course studies, thrombin promoted a significantly greater phosphorylation of the 20- and 47 KDa proteins in platelets of SHR compared with that for WKY. Sensitivity of myosin light chain kinase to the calcium ionophore A23187 and of protein kinase C to both phorbol ester and dioctanoylglycerol was apparently not different between the two cell types. The data indicate that the exaggerated thrombin-induced protein phosphorylation observed for platelets of SHR is not linked to alterations in protein kinase C and/or myosin light chain kinase per se. These results therefore suggest that platelet hyperresponsiveness in SHR is likely to be related, at least in part, to abnormalities in receptor-mediated transmembrane signalling.


Subject(s)
Blood Platelets/metabolism , Hypertension/blood , Phosphoproteins/blood , Animals , Blood Platelets/drug effects , Male , Myosin-Light-Chain Kinase/blood , Protein Kinase C/blood , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Receptors, Cell Surface/drug effects , Receptors, Cell Surface/physiology , Thrombin/pharmacology
14.
J Biol Chem ; 264(15): 8575-9, 1989 May 25.
Article in English | MEDLINE | ID: mdl-2722789

ABSTRACT

We examined the extranuclear effects of thyroid hormones on human platelets. Pretreatment with DL-thyroxine or DL-triiodothyronine inhibited collagen-induced aggregation, in a dose-dependent manner, but other derivatives of thyroid hormone had no significant effects. In contrast to collagen, 12-O-tetradecanoylphorbol-13-acetate-induced aggregation was not affected by thyroid hormones at the same concentration range. Thyroxine also inhibited the release of [14C] serotonin from collagen-stimulated platelets, with a marked reduction in the phosphorylation of 20,000-dalton protein. Thyroxine and triiodothyronine had inhibitory effects on myosin light chain kinase purified from human platelets and inhibited more markedly the myosin light chain kinase than protein kinase C (Ca2+/phospholipid-dependent enzyme) and cAMP-dependent protein kinase. In addition, L-thyroxine behaved as a competitive inhibitor of myosin light chain kinase toward calmodulin, and the Ki value was calculated to be 2.6 microM. To determine whether or not thyroxine directly binds myosin light chain kinase, we prepared an affinity column, using L-thyroxine as the ligand. Myosin light chain kinase was selectively bound to the column while calmodulin passed through. We also designed a procedure for the purification of myosin light chain kinase from human platelets, using L-thyroxine-affinity chromatography. A markedly increased purification was thus achieved, and DEAE-cellulose and L-thyroxine-affinity chromatography were made feasible. These results suggest that thyroxine can serve as a pharmacological tool for elucidating the biological significance of myosin light chain kinase-mediated reactions and is a pertinent ligand which can be used to purify myosin light chain kinase from platelets as a substitute for calmodulin.


Subject(s)
Blood Platelets/physiology , Myosin-Light-Chain Kinase/blood , Platelet Aggregation Inhibitors , Platelet Aggregation/drug effects , Thyroxine/pharmacology , Triiodothyronine/pharmacology , Tyrosine/pharmacology , Blood Platelets/drug effects , Chromatography, Affinity , Chromatography, DEAE-Cellulose , Humans , Kinetics , Molecular Weight , Myosin-Light-Chain Kinase/antagonists & inhibitors , Myosin-Light-Chain Kinase/isolation & purification , Protein Kinase C/blood , Protein Kinases/blood , Serotonin/blood
15.
J Am Coll Cardiol ; 11(3): 487-93, 1988 Mar.
Article in English | MEDLINE | ID: mdl-3343452

ABSTRACT

To detect myocardial cell damage, serum samples of 42 consecutive patients with angina at rest were screened for cardiac myosin light chains, which were detected in 22 patients (52%). In 17 of these patients there was a persistent release of myosin light chains lasting until the 4th hospital day, whereas in 7 patients myosin light chains were only detectable during the initial 24 h after admission. The presence of myosin light chains correlated with signs of ischemia in the electrocardiogram (ECG) (p less than 0.05) and with the extent of coronary artery narrowing (p less than 0.05). Cardiac myosin light chains were elevated in serum only if there was a greater than or equal to 75% diameter narrowing in at least one major vessel. In all five patients who developed transmural myocardial infarction during the course of their hospital stay, myosin light chains were detectable greater than or equal to 28 h before the diagnosis of myocardial infarction could be established by ECG criteria and conventional serum enzymes. Thus the detection of circulating cardiac myosin light chains enables one to identify a subgroup of patients with angina at rest having more severe coronary artery disease with a worse outcome.


Subject(s)
Angina Pectoris, Variant/enzymology , Myocardium/enzymology , Myosin-Light-Chain Kinase/blood , Adult , Aged , Angina Pectoris, Variant/diagnostic imaging , Angina Pectoris, Variant/physiopathology , Coronary Angiography , Electrocardiography , Female , Humans , Male , Middle Aged , Myocardial Infarction/blood , Prognosis , Rest , Risk Factors , Time Factors
16.
Biochem J ; 249(2): 345-50, 1988 Jan 15.
Article in English | MEDLINE | ID: mdl-3257691

ABSTRACT

The ability of several putative inhibitors of protein kinase C (PKC) to block dioctanoylglycerol (DC8)-induced phosphorylation of a 47 kDa protein (a recognized substrate for PKC) in human platelets was investigated. Staurosporine (1 microM) caused complete inhibition of phosphorylation, whereas the other reagents were either inactive (polymyxin B) or gave only partial inhibition (C-1, H-7, tamoxifen). Staurosporine (1 microM) fully inhibited the phosphorylation of the 47 kDa protein in platelets challenged with thrombin, but also inhibited the phosphorylation of a 20 kDa protein which is a substrate for myosin light-chain kinase. The inhibition of both kinases by staurosporine was associated with the inhibition of thrombin-induced secretion of ATP and 5-hydroxytryptamine and a slowing of the aggregation response; staurosporine, however, had no effect on the formation of phosphatidic acid and inositol phosphates induced by thrombin. Staurosporine also reversed the inhibitory action of phorbol esters on thrombin-induced formation of phosphatidic acid. These data are consistent with a role for these two kinases in secretion and aggregation (although there must be additional control signals, since aggregation was only slowed, not inhibited), but suggest that neither kinase is involved in the regulation of phosphoinositide metabolism. This latter conclusion contradicts previous observations that the activation of PKC by phorbol esters or membrane-permeable diacylglycerols alters the apparent activity of both phospholipase C and inositol trisphosphatase. Possible explanations for this discrepancy are discussed.


Subject(s)
Alkaloids/pharmacology , Blood Platelets/metabolism , Inositol Phosphates/biosynthesis , Protein Kinase C/antagonists & inhibitors , Sugar Phosphates/biosynthesis , Thrombin/pharmacology , Adenosine Triphosphate/blood , Blood Platelets/drug effects , Blood Proteins/metabolism , Humans , Inositol 1,4,5-Trisphosphate , Inositol Phosphates/blood , Myosin-Light-Chain Kinase/antagonists & inhibitors , Myosin-Light-Chain Kinase/blood , Phosphorylation , Protein Kinase C/blood , Serotonin/blood , Staurosporine , Type C Phospholipases/blood
17.
Circulation ; 76(6): 1251-61, 1987 Dec.
Article in English | MEDLINE | ID: mdl-3677350

ABSTRACT

To estimate the extent of myocardial infarction after coronary artery reperfusion, serum levels of cardiac myosin light chain (LC) I and creatine kinase (CK) were determined serially in 49 patients with acute myocardial infarction. Intracoronary thrombolysis was successful in 25 patients (reperfusion group), and 24 patients were treated in a conventional manner (control group). The peak level of CK appeared significantly earlier in the reperfusion group (11.3 +/- 3.1 hr, mean +/- SD) than in the control group (21.6 +/- 7.2 hr). Cumulative release of CK was significantly related to angiographically determined left ventricular ejection fraction 1 month after the attack in both groups (r = -.50; -.45, respectively). However, the amount of cumulative release of CK in the reperfusion group was greater compared with that in those with the same left ventricular ejection fraction in the control group. Peak appearance time of LCI was almost equal in the two groups (3.8 +/- 1.4 vs 3.9 +/- 1.2 days). Peak levels of LCI were related to the left ventricular ejection fraction in the reperfusion group (r = -.63) and in the control group (r = -.74), and the slopes of their regression lines were similar. The cardiac index obtained on the day of onset in the two groups was related to peak levels of LCI but not to total release of CK. These results suggest that serum levels of LCI reflect the changes in left ventricular function after acute myocardial infarction, regardless of the presence of coronary reperfusion. Thus, serial determinations of LCI in serum facilitate noninvasive assessment of the effects of intracoronary thrombolysis on infarct size.


Subject(s)
Fibrinolytic Agents/administration & dosage , Heart/physiopathology , Myocardial Infarction/drug therapy , Myocardium/enzymology , Myosin-Light-Chain Kinase/blood , Adult , Aged , Clinical Enzyme Tests , Coronary Vessels , Creatine Kinase/blood , Female , Humans , Male , Middle Aged , Myocardial Infarction/diagnosis , Myocardial Infarction/physiopathology , Urokinase-Type Plasminogen Activator/administration & dosage
18.
Am J Physiol ; 253(5 Pt 2): H1035-43, 1987 Nov.
Article in English | MEDLINE | ID: mdl-3688248

ABSTRACT

The specific effects of U 46619 (9,11-dideoxy,9 alpha-11 alpha-methanoepoxyprostaglandin F2 alpha), thromboxane A2-prostaglandin H2 (TxA2/PGH2) analogue, on human platelet shape change, myosin light-chain phosphorylation, serotonin release, fibrinogen receptor exposure, and platelet aggregation were measured and compared with binding of [3H]U 46619 to platelets. Shape change and myosin light-chain phosphorylation were found to be saturable and dose dependent, having effective concentration producing 50% of the maximum response (EC50) values of 0.035 +/- 0.005 and 0.057 +/- 0.021 microM, respectively (mean +/- SE). These two effects were competitively inhibited by specific antagonists of TxA2/PGH2 receptors (BM 13177, PTA-OH, and 1.PTA-OH) indicating that they are receptor mediated. Binding of [3H]U 46619 showed two components. Occupancy of high-affinity binding sites [dissociation constant (Kd) = 0.041 +/- 0.009 microM, maximum binding site (Bmax) = 19.4 +/- 5.3 fmol/10(7) platelets, with 1,166 +/- 310 sites/platelet; n = 12] correlated with platelet shape change and myosin light-chain phosphorylation. We propose that a second component with an apparent Kd of 1.46 +/- 0.47 microM (n = 12) represents a second, low-affinity site. Mean EC50 values for U 46619-induced serotonin release, platelet aggregation, and fibrinogen receptor exposure were 0.54 +/- 0.13. 1.31 +/- 0.34 and 0.53 +/- 0.21 microM, respectively. Therefore, the platelet release reaction was not directly correlated with occupancy of high-affinity receptors but could be related to the second binding component of U 46619. Fibrinogen receptor exposure and platelet aggregation caused by U 46619 appeared to be events mediated by the release of adenosine diphosphate from platelet-dense granules.


Subject(s)
Blood Platelets/drug effects , Prostaglandin Endoperoxides, Synthetic/pharmacology , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid , Adenosine Diphosphate/blood , Humans , Kinetics , Myosin-Light-Chain Kinase/blood , Platelet Aggregation Inhibitors/pharmacology , Platelet Membrane Glycoproteins/metabolism , Serotonin/blood , Sulfonamides/pharmacology
19.
Biochem Int ; 13(4): 571-8, 1986 Oct.
Article in English | MEDLINE | ID: mdl-3026398

ABSTRACT

We previously demonstrated that myosin light chain kinase (MLCK) of gizzard is proteolyzed by platelet calpain. It has been also reported that partially cleaved MLCK may phosphorylate myosin light chain (20K) in the absence of calmodulin. Therefore, a possible participation of calpain in 20K phosphorylation was studied in human platelets, utilizing various inhibitors. An epoxy succinate derivative (E-64) or N-ethylmaleimide (NEM), used as calpain antagonist, inhibited 20K phosphorylation of Ca2+-stimulated lysed platelets. A synergistic effect between these calpain antagonists and calmodulin antagonist W-7 was observed. Also, the similar results were obtained in 20K phosphorylation of intact platelets. From these observations, it was suggested that 20K phosphorylation in platelets is mediated by two separate pathways, namely calmodulin and calpain dependent pathways, provided that calpain activity is specifically inhibited by the antagonists used.


Subject(s)
Blood Platelets/metabolism , Calpain/blood , Myosin-Light-Chain Kinase/blood , Myosins/blood , Peptide Fragments/blood , Ca(2+) Mg(2+)-ATPase/blood , Humans , Kinetics , Myosin Subfragments , Phosphorylation , Thrombin/physiology
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