1.Effects of calmodulin-dependent protein kinase II inhibitor, KN-93, on electrophysiological features of rabbit hypertrophic cardiac myocytes.
Jun KE ; Feng CHEN ; Cuntai ZHANG ; Xing XIAO ; Jing TU ; Musen DAI ; Xiaoping WANG ; Bing CHEN ; Min CHEN
Journal of Huazhong University of Science and Technology (Medical Sciences) 2012;32(4):485-489
Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related. The aim of this study was to determine the effects of the calmodulin-dependent protein kinase (CaMK) II inhibitor, KN-93, on L-type calcium current (I(Ca, L)) and early after-depolarizations (EADs) in hypertrophic cardiomyocytes. A rabbit model of myocardial hypertrophy was constructed through abdominal aortic coarctation (LVH group). The control group (sham group) received a sham operation, in which the abdominal aortic was dissected but not coarcted. Eight weeks later, the degree of left ventricular hypertrophy (LVH) was evaluated using echocardiography. Individual cardiomyocyte was isolated through collagenase digestion. Action potentials (APs) and I(Ca, L) were recorded using the perforated patch clamp technique. APs were recorded under current clamp conditions and I(Ca, L) was recorded under voltage clamp conditions. The incidence of EADs and I(ca, L) in the hypertrophic cardiomyocytes were observed under the conditions of low potassium (2 mmol/L), low magnesium (0.25 mmol/L) Tyrode's solution perfusion, and slow frequency (0.25-0.5 Hz) electrical stimulation. The incidence of EADs and I(ca, L) in the hypertrophic cardiomyocytes were also evaluated after treatment with different concentrations of KN-92 (KN-92 group) and KN-93 (KN-93 group). Eight weeks later, the model was successfully established. Under the conditions of low potassium, low magnesium Tyrode's solution perfusion, and slow frequency electrical stimulation, the incidence of EADs was 0/12, 11/12, 10/12, and 5/12 in sham group, LVH group, KN-92 group (0.5 μmol/L), and KN-93 group (0.5 μmol/L), respectively. When the drug concentration was increased to 1 μmol/L in KN-92 group and KN-93 group, the incidence of EADs was 10/12 and 2/12, respectively. At 0 mV, the current density was 6.7±1.0 and 6.3±0.7 PA·PF(-1) in LVH group and sham group, respectively (P>0.05, n=12). When the drug concentration was 0.5 μmol/L in KN-92 and KN-93 groups, the peak I(Ca, L) at 0 mV was decreased by (9.4±2.8)% and (10.5±3.0)% in the hypertrophic cardiomyocytes of the two groups, respectively (P>0.05, n=12). When the drug concentration was increased to 1 μmol/L, the peak I(Ca, L) values were lowered by (13.4±3.7)% and (40±4.9)%, respectively (P<0.01, n=12). KN-93, a specific inhibitor of CaMKII, can effectively inhibit the occurrence of EADs in hypertrophic cardiomyocytes partially by suppressing I(Ca, L), which may be the main action mechanism of KN-93 antagonizing the occurrence of ventricular arrhythmias in hypertrophic myocardium.
Animals
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Benzylamines
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pharmacology
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Calcium-Calmodulin-Dependent Protein Kinase Type 2
;
antagonists & inhibitors
;
metabolism
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Cardiomegaly
;
metabolism
;
physiopathology
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Female
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Myocytes, Cardiac
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drug effects
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metabolism
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physiology
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Rabbits
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Sulfonamides
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pharmacology
2.Effects of niflumic acid on the proliferation of human hepatoma cells.
Jing TIAN ; Ling TAO ; Yun-Xin CAO ; Ling DONG ; Yu-Zhen HU ; An-Gang YANG ; Shi-Sheng ZHOU
Acta Physiologica Sinica 2003;55(2):160-164
The purpose of this work was to investigate the effects of niflumic acid (NFA), a chloride channel blocker, on the proliferation of human hepatoma cell line (HHCC). Cell proliferation was analyzed by cell count and MTT assay. Cell cycle analysis was carried out by flow cytometry. [Ca(2+)](i) was determined by laser scanning confocal system. It was found that NFA decreased significantly the cell number and the MTT optical density (OD) of HHCC cells, and that the OD value was reversed after washout of NFA. Compared with control, NFA blocked cell cycle progression in G(1) phase. Extracellular application of NFA (100 micromol/L) induced a rapid decrease in [Ca(2+)](i). These findings demonstrate that blockage of chloride channels by NFA induces growth arrest of HHCC in G(1) phase, which may be due to the inhibition of Ca(2+)/CaM-dependent signaling pathways.
Calcium
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metabolism
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Calmodulin
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metabolism
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Carcinoma, Hepatocellular
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pathology
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Cell Line, Tumor
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Cell Proliferation
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drug effects
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Chloride Channels
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antagonists & inhibitors
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Humans
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Liver Neoplasms
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pathology
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Niflumic Acid
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pharmacology
3.Effect of calmodulin antagonist O-4-ethoxyl- butyl-berbamine on the proliferation of human breast cancer cell line MCF-7 and its relevant mechanism.
Xiao-Hua DAI ; Rong LIU ; Yuan ZHOU ; Ming YANG ; Dong-Mei FAN ; Dong-Sheng XIONG ; Jing QI ; Chun-Zheng YANG ; Hui-Fang ZHU
Acta Academiae Medicinae Sinicae 2009;31(3):326-329
OBJECTIVETo investigate the effect of calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine (EBB) on proliferation of human breast cancer cell MCF-7 and its possible mechanism.
METHODSMTT assay was used to analyze the effect of EBB on tumor cells growth. Flow cytometry was used to detect its impact on the cell cycle of MCF-7 cells. Immunofluoresce labeling technique and laser scanning confocal microscope were used to reveal the changes of the microtubule, microfilament, mitochondrion, and endoplasmic reticulum in the cells.
RESULTSThe IC50 value of EBB in MCF-7 cells was (13.0 +/- 3.7) micromol/L. MCF-7 cells were arrested at S phase after EBB treatment. Meanwhile, depolymerization of the microtubule and microfilament, impairment of the mitochondrion and swelling of endoplasmic reticulum were observed.
CONCLUSIONEBB arrests MCF-7 cells at S phase by inhibiting the growth of MCF-7 cells, which may be related to the changes of structures and functions of the microtubule, microfilament, mitochondrion, and endoplasmic reticulum.
Benzylisoquinolines ; pharmacology ; Breast Neoplasms ; pathology ; Calmodulin ; antagonists & inhibitors ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Female ; Humans
4.Role of calmodulin-dependent protein kinase II in bupivacaine hydrochloride-induced injury of SH-SY5Y cells.
Xianjie WEN ; Jiying ZHONG ; Tao ZHANG ; Xiaohong LAI ; Hongzhen LIU ; Hanbing WANG ; Chengxiang YANG
Journal of Southern Medical University 2015;35(8):1133-1136
OBJECTIVETo investigate the effect of KN93, a calmodulin-dependent protein kinase II (CaMK II) inhibitor, on SH-SY5Y cell injury induced by bupivacaine hydrochloride.
METHODSSH-SY5Y cells exposed for 24 h to 1 mmol/L KN93, 1 mmol/L bupivacaine hydrochloride, or both were examined for morphological changes and Cav3.1 protein expressions using Western blotting. The vitality and apoptosis rate of the cells at different time points during the exposures were assessed with MTT assay and flow cytometry, respectively.
RESULTSBupivacaine hydrochloride exposure caused obvious cell morphologial changes, reduced cell viability, increased cell apoptosis, and enhanced Cav3.1 protein expression. All these changes were partly reversed by treatment of the cells with 1 mmol/L KN93.
CONCLUSIONSCaMKII may play a role in bupivacaine hydrochloride-induced SH-SY5Y cells injury, which is related with upregulated Cav3.1 protein expression.
Apoptosis ; Bupivacaine ; adverse effects ; Calcium Channels, T-Type ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; antagonists & inhibitors ; metabolism ; Cell Line ; Cell Survival ; Humans ; Up-Regulation
5.Calmodulin antagonist inhibits torsade de pointes induced by d-sotalol in an isolated rabbit heart model.
Jun PU ; Cun-tai ZHANG ; Rong BAI ; Nian LIU ; Yang LI ; Lin WANG
Chinese Journal of Cardiology 2005;33(4):364-368
OBJECTIVETo evaluate the effects of W-7, a calmodulin inhibitor, on transmural dispersion of repolarization (TDR), early after depolarization (EAD) and torsade de pointes (TdP) induction after administration of d-sotalol in isolated rabbit heart.
METHODSTdP was induced by d-sotalol (30 micromol/L), bradycardia, and hypokalemic (1.5 mmol/L)/hypomagnesaemic (0.35 mmol/L) solution in isolated female rabbit hearts. Thirty six rabbit hearts were divided into 4 groups (n = 9 each): d-sotalol alone, d-sotalol + W-7 (20 micromol/L), d-sotalol + W-7 (50 micromol/L), and d-sotalol + W-7 (100 micromol/L). Monophasic action potentials (MAPs) of the left ventricular epimyocardium (Epi), midmyocardium (M), and endomyocardium (Endo) were recorded simultaneously with ECG. The incidence of EAD and TdP were observed as well.
RESULTSTreatment with d-sotalol alone prolonged ventricular MAP duration and QT interval, increased TDR, and evoked high incidence of EAD (9/9) and spontaneous TdP (7/9) in hypokalemic/hypomagnesaemic solution in female rabbit heart. W-7 concentration-dependently decreased incidence of TdP (4/9 in 20 micromol/L; 2/9 in 50 micromol/L; 1/9 in 100 micromol/L). This effect of W-7 coincided with the decreased incidence of EAD (5/9 in 20 micromol/L; 4/9 in 50 micromol/L; 1/9 in 100 micromol/L). However, the d-sotalol-induced prolongation of QT interval and TDR was not significantly altered by W-7 at the three concentration used.
CONCLUSIONSIn isolated female rabbit hearts, calmodulin antagonist W-7 suppresses d-sotalol-induced TdP without altering TDR but does suppress EAD. The effects observed with W-7 also suggest a possible important role for calmodulin-activated enzymes in the induction of TdP.
Animals ; Calmodulin ; antagonists & inhibitors ; Enzyme Inhibitors ; therapeutic use ; Female ; In Vitro Techniques ; Rabbits ; Sotalol ; adverse effects ; Sulfonamides ; therapeutic use ; Torsades de Pointes ; chemically induced ; prevention & control
6.Volatile anesthetics inhibit the activity of calmodulin by interacting with its hydrophobic site.
Miao-Miao ZHOU ; Hui-Min XIA ; Jiao LIU ; You-Nian XU ; Nai-Xin XIN ; Shi-Hai ZHANG
Chinese Medical Journal 2012;125(17):3166-3170
BACKGROUNDVolatile anesthetics (VAs) may affect varied and complex physiology processes by manipulating Ca(2+)-calmodulin (CaM). However, the detailed mechanism about the action of VAs on CaM has not been elucidated. This study was undertaken to examine the effects of VAs on the conformational change, hydrophobic site, and downstream signaling pathway of CaM, to explore the possible mechanism of anesthetic action of VAs.
METHODSReal-time second-harmonic generation (SHG) was performed to monitor the conformational change of CaM in the presence of VAs, each plus 100 µmol/L Ca(2+). A hydrophobic fluorescence indicator, 8-anilinonaphthalene-1-sulfonate (ANS), was utilized to define whether the VAs would interact with CaM at the hydrophobic site or not. High-performance liquid chromatography (HPLC) was carried out to analyze the activity of CaM-dependent phosphodiesterase (PDE1) in the presence of VAs. The VAs studied were ether, enflurane, isoflurane, and sevoflurane, with their aqueous concentrations 7.6, 9.5, 11.4 mmol/L; 0.42, 0.52, 0.62 mmol/L; 0.25, 0.31, 0.37 mmol/L and 0.47, 0.59, 0.71 mmol/L respectively, each were equivalent to their 0.8, 1.0 and 1.2 concentration for 50% of maximal effect (EC50) for general anesthesia.
RESULTSThe second-harmonic radiation of CaM in the presence of Ca(2+) was largely inhibited by the VAs. The fluorescence intensity of ANS, generated by binding of Ca(2+) to CaM, was reversed by the VAs. HPLC results also showed that AMP, the product of the hydrolysis of cAMP by CaM-dependent PDE1, was reduced by the VAs.
CONCLUSIONSOur findings demonstrate that the above VAs interact with the hydrophobic core of Ca(2+)-CaM and the interaction results in the inhibition of the conformational change and activity of CaM. This in vitro study may provide us insight into the possible mechanism of anesthetic action of VAs in vivo.
Adenosine Monophosphate ; analysis ; Anesthetics, Inhalation ; pharmacology ; Anilino Naphthalenesulfonates ; Calmodulin ; antagonists & inhibitors ; chemistry ; physiology ; Cyclic Nucleotide Phosphodiesterases, Type 1 ; analysis ; Fluorescence ; Humans ; Hydrophobic and Hydrophilic Interactions
7.The Regulation of AP-1 DNA Binding Activity by Long-term Nicotine Stimulation in Bovine Adrenal Medullary Chromaffin Cells: Role of Second Messengers.
Jin Koo LEE ; Seong Soo CHOI ; Hong Won SUH
The Korean Journal of Physiology and Pharmacology 2002;6(2):109-112
The signal pathways involved in the regulation of AP-1 DNA binding activity in long-term nicotine stimulated bovine adrenal medullary chromaffin (BAMC) cells have not been well characterized. To understand the involvement of second messengers in the regulation of AP-1 DNA binding activity, the present study was designed to define the time-course for inhibition of nicotine-induced responses by cholinergic antagonists, Ca2+ and calmodulin (CaM) antagonists, and calcium/calmodulin-dependent protein kinase (CaMK) II inhibitor using electrophoretic mobility shift assay. Nicotine (10microM) stimulation increased AP-1 DNA binding activity at 24 hr after treatment. Posttreatment with hexamethonium (1 mM) plus atropine (1microM) (HA), nimodipine (1microM), or calmidazolium (1microM) at 0.5, 3, and 6 hr after the nicotine treatment significantly inhibited the AP-1 DNA binding activity increased by long-term nicotine stimulation. However, posttreatment with HA, nimodipine, or calmidazolium at 9 or 12 hr after the nicotine treatment did not affect the nicotine-induced increase of AP-1 DNA binding activity. The pretreatment of BAMC cells with various concentrations of KN-62 inhibited the increase of AP-1 DNA binding activity induced by nicotine in a concentration-dependent manner. KN-62 (10microM) posttreatment beginning at 0.5, 3, or 6 hr after the nicotine treatment significantly inhibited the increase of AP-1 DNA binding activity. However, KN-62 posttreatment beginning at 9 or 12 hr after the nicotine treatment did not affect the increase of AP-1 DNA binding activity. This study suggested that stimulation (for at least 6 hr) of nicotinic receptors on BAMC cells was necessary for increase of AP-1 DNA binding activity, and activation of Ca2+, CaM, and CaMK II up to 6 hr at least seemed to be required for the increase of nicotine-induced AP-1 DNA binding activity.
Atropine
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Calmodulin
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Cholinergic Antagonists
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Chromaffin Cells*
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DNA*
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Electrophoretic Mobility Shift Assay
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Hexamethonium
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Nicotine*
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Nimodipine
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Protein Kinases
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Receptors, Nicotinic
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Second Messenger Systems*
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Signal Transduction
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Transcription Factor AP-1*
8.Effects of calcium and calmodulin dependent kinase against hypoxic neuronal injury.
Hui ZHOU ; Xiao-Mei SUN ; Xiao-Li LUO ; Meng MAO
Chinese Journal of Contemporary Pediatrics 2007;9(4):324-326
OBJECTIVETo study the effects of calcium and calmodulin dependent kinase against hypoxic neuronal injury and its possible mechanisms.
METHODSEmbryonic cortical neurons of 17-day pregnant embryo Sprague-Dawley rats were cultured in vitro and the cultured neurons were randomly allocated into different groups that exposed to hypoxia or hypoxia +calcium channel antagonist. Nimodipine and MK-801 were used to block the L-voltage sensitive calcium channel and NMDA receptor respectively before hypoxia. The methyl thiazolyl tetrazolium (MTT) method was used to determine the cell viability. Fluo-4AM, an intracellular calcium indictor, was used to detect the changes of intracellular calcium after hypoxia. The expressions of CaMKII and CaMKIV were detected by Western blot.
RESULTSThe cell viability of the nimodipine or MK-801-treated groups was significantly higher than that of the untreated hypoxia group. The intracellular calcium level of the nimodipine-treated group decreased rapidly after hypoxia. Compared to nimodipine treatment, MK-801 treatment could inhibit hypoxia-induced calcium influx for a longer time. Nimodipine treatment decreased the CaMKII expression while MK-801 treatment decreased the CaMKIV expression.
CONCLUSIONSNimodipine and MK-801 protect neurons from hypoxic injury possibly by the inhibition of CaMKII and CaMKIV expressions respectively.
Animals ; Calcium ; analysis ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinase Type 4 ; Calcium-Calmodulin-Dependent Protein Kinases ; antagonists & inhibitors ; physiology ; Cell Hypoxia ; Dizocilpine Maleate ; pharmacology ; Female ; Neurons ; pathology ; Neuroprotective Agents ; pharmacology ; Nimodipine ; pharmacology ; Rats ; Rats, Sprague-Dawley
9.Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system.
Yoshitaka FUKAYA ; Masayuki KURODA ; Yasuyuki AOYAGI ; Sakiyo ASADA ; Yoshitaka KUBOTA ; Yoshitaka OKAMOTO ; Toshinori NAKAYAMA ; Yasushi SAITO ; Kaneshige SATOH ; Hideaki BUJO
Experimental & Molecular Medicine 2012;44(5):330-339
Auto-transplantation of adipose tissue is commonly used for the treatment of tissue defects in plastic surgery. The survival of the transplanted adipose tissue is not always constant, and one of reasons is the accelerated apoptosis of the implanted preadipocytes. We have recently established highly homogeneous preadipocytes, named ccdPAs. The aim of the current study was to evaluate the regulation of the potency of platelet-rich plasma (PRP) on the apoptosis of ccdPAs in vitro. PRP stimulated the proliferation of the preadipocytes in a dose-dependent manner, and the stimulatory activity of 2% PRP was significantly higher than that of 2% FBS or 2% platelet-poor plasma (PPP). The presence of 2% PRP significantly inhibited serum starvation- or TNF-alpha/cycloheximide-induced apoptosis in comparison to 2% FBS or 2% PPP. DAPK1 and Bcl-2-interacting mediator of cell death (BIM) mRNAs were reduced in the preadipocytes cultured with 2% PRP in comparison to those cultured in 2% FBS. The gene expression levels were significantly higher in cells cultured without serum in comparison to cells cultured with 2% FBS, and the levels in the cells with 2% PRP were reduced to 5-10% of those in the cells without serum. These results indicated that ccdPAs exhibit anti-apoptotic activities, in addition to increased proliferation, when cultured in 2% PRP in comparison to the same concentration of FBS, and that this was accompanied with reduced levels of DAPK1 and BIM mRNA expression in in vitro culture. PRP may improve the outcome of transplantation of adipose tissue by enhancing the anti-apoptotic activities of the implanted preadipocytes.
Adipocytes/*cytology
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Adipose Tissue/cytology/metabolism
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Apoptosis/*physiology
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Apoptosis Regulatory Proteins/antagonists & inhibitors/metabolism
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Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors/metabolism
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Cell Culture Techniques/*methods
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*Cell Differentiation
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Cell Proliferation
;
Cells, Cultured
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Gene Expression Regulation
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Humans
;
Membrane Proteins/antagonists & inhibitors/metabolism
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*Platelet-Rich Plasma/metabolism/physiology
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Proto-Oncogene Proteins/antagonists & inhibitors/metabolism
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Tissue Transplantation
10.Influence of calcium ion on host cell invasion and intracellular replication by Toxoplasma gondii.
Hyun Ouk SONG ; Myoung Hee AHN ; Jae Sook RYU ; Duk Young MIN ; Kyoung Hwan JOO ; Young Ha LEE
The Korean Journal of Parasitology 2004;42(4):185-193
Toxoplasma gondii is an obligate intracellular protozoan parasite, which invades a wide range of hosts including humans. The exact mechanisms involved in its invasion are not fully understood. This study focused on the roles of Ca2+ in host cell invasion and in T. gondii replication. We examined the invasion and replication of T. gondii pretreated with several calcium modulators, the conoid extrusion of tachyzoites. Calmodulin localization in T. gondii were observed using the immunogold method, and Ca2+ levels in tachyzoites by confocal microscopy. In light microscopic observation, tachyzoites co-treated with A23187 and EGTA showed that host cell invasion and intracellular replication were decreased. The invasion of tachyzoites was slightly inhibited by the Ca2+ channel blockers, bepridil and verapamil, and by the calmodulin antagonist, calmidazolium. We observed that calcium saline containing A23187 induced the extrusion of tachyzoite conoid. By immunoelectron microscopy, gold particles bound to anti-calmodulin or anti-actin mAb, were found to be localized on the anterior portion of tachyzoites. Remarkably reduced intracellular Ca2+ was observed in tachyzoites treated with BAPTA/AM by confocal microscopy. These results suggest that host cell invasion and the intracellular replication of T. gondii tachyzoites are inhibited by the calcium ionophore, A23187, and by the extracellular calcium chelator, EGTA.
Animals
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Calcium/*physiology
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Calcium Channel Blockers/pharmacology
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Calmodulin/antagonists & inhibitors
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Chelating Agents/pharmacology
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Hela Cells
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Host-Parasite Relations
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Humans
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Ionophores/pharmacology
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Research Support, Non-U.S. Gov't
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Toxoplasma/drug effects/pathogenicity/*physiology