Effect of Ba2+ concentration on L-type Ca2+ channel in hypothalamic neurons

AIM:To investigate the effect of Ba²⁺ concentration on L-type of Ca²⁺ channel in hypothalamic neurons.METHODS:The cell acute isolation technique and cell-attached patch-clamp technique were used.RESULTS:The slope conductance of L-type Ca²⁺ channel were 28.6 pS (110 mmol/L) and 19.1 pS (10 mmol/L), and the open probability (NP₀) obviously different with different Ba²⁺ concentration as carrier. CONCLUSION:Ba²⁺ concentration had the obvious effect on the L-type Ca²⁺ channel.     

Effects of hydrogen peroxide on sodium current in acutely isolated rat hippocampal CA1 neurons

AIM and METHODS: The effects of hydrogen peroxide on Na⁺ currents were studied in freshly dissociated rat hippocampal CA1 neurons using the whole-cell patch-clamp techinique. RESULTS: ①H₂O₂ caused a dose-dependent and voltage-dependent increase in the voltage-activated Na⁺ currents. The amplitudes of Na⁺ currents were increased (48.0±4.2)% and (88. 2±5. 1)% (n=10) by H₂O₂ at 10 μmol/L and 100 μmol/L, respectively. ②H₂O₂ (10 μmol/L) did not affect the activation process, but changed the inactivation process significantly. Before and after application of 10 μmol/L of H₂O₂, the half-inactivation voltage was (-64.58±1.22)mV and (-53.55±0.94)mV (n=10, P<0.01), but the slope factor was not changed. CONCLUSION: As a product of oxidation metabolism, H₂O₂ is related to some diseases in the central nervous system.     

Hypotonic challenges induce volume-activated chloride currents and regulatory volume decrease in rat embryonic myocardial H9c2 cells

AIM:To investigate the volume-activated chloride currents and regulatory volume decrease(RVD) induced by hypotonic challenges in rat embryonic myocardial cell line H9c2. METHODS:The technique of whole-cell patch-clamp was used to record the chloride currents induced by hypotonic challenges and to clarify the properties of the currents in H9c2 cells. The changes of cell volume were observed by the technique of real-time living cell imaging, and the roles of chloride channels in RVD were analyzed. RESULTS:A weak background current was recorded in H9c2 cells under isotonic condition. Extracellular application of 47% hypotonic solution rapidly activated an outward rectified current, which did not exhibit time-and voltage-dependent inactivation with the current density of(47.77±3.80) pA/pF at +80 mV and(-33.36±2.80) pA/pF at-80 mV. The reversal potential was(-9.02±0.61) mV, closed to the calculated equilibrium potential for Cl^-(-0.9 mV). The current was volume-sensitive and was completely suppressed by 47% hypertonic solution. In addition, chloride channel blockers tamoxifen(20 μmol/L), 5-nitro-2-(3-phenylpropylamino) benzoic acid(NPPB,100 μmol/L) and ATP(10 mmol/L) significantly inhibited the current with different inhibitory ratios. The phenomenon of RVD was also observed in H9c2 cells under the condition of perfusion with 47% hypotonic solution. The chloride channel blocker NPPB at concentration of 100 μmol/L completely inhibited the RVD process. CONCLUSION:The volume-activated chloride channels, which are activated by extracellular hypotonic challenges, play an important role in the process of regulatory volume decrease in H9c2 cells.     

Effects of glucose and Mg2+ in the neurons damaged by glutamate

AIM and METHODS: To observe the effects of glucose-free and Mg²⁺-free in the extracellular fluid on the changes of [Ca ²⁺]_i in the cerebro-cortical neurons damaged by 1mmol/L glutamate using laser confocal scanning microscope. RESULTS: Both frequency and amplitude of neuronal calcium oscillation induced by glutamate were lowered in glucose-free and Mg²⁺-free buffers. The basic [Ca²⁺]_i concentration was lowered in the former case , but it was elevated in the latter case. CONCLUSION: Mg²⁺-free aggravates [Ca²⁺]_i overload induced by 1mmol/L glutamate ,under certain conditions the glucose-free might resist damage role of glutamate and Mg²⁺-free.     

Role of calcium ion in the cyotoxicity and DNA fragment induction in HL-60 cells by 6F isolated from Pteris semipinnata L.

AIM: To investigate the changes of cytosolic free calcium concentration([Ca²⁺]_i) and expression of Bcl-2 in HL-60 cells treated by 6F isolated from Pteris semipinnata L.(PSL), and to discuss the relations between calcium ion and cytotoxicity and DNA fragment induction effects of 6F. METHODS: HL-60 cells were used as in vitro model. [Ca²⁺]_i was measured on fluorescent spectrophotometry using Fura-2/AM as Ca²⁺ indicator. Bcl-2 expressing level was measured by flow cytometry. Tetrazolium salt(MTT) and diphenylamine staining methods were applied for cytotoxicity assay and DNA fragmentation detection, respectively. RESULTS: [Ca²⁺]_i increased obviously in a dose and time dependent manner after treated HL-60 cells with 6F. 6F decreased the expressing level of Bcl-2. Adding 2 mmol/L Ca²⁺ to the medium, or 1 mmol/L EDTA to chelate Ca²⁺, or 4 μmol/L calcium ionophore A 23187 to increase the concentration of cytosolic Ca²⁺, the DNA fragment induction by 6F was not affected, whereas the cytotoxicity of 6F was enhanced. 250 μmol/L Zn²⁺ attenuated the DNA fragment induction, and the cytotoxicity of 6F against HL-60 cells was enhanced significantly. CONCLUSION: It was speculated that the decreased expressing of Bcl-2 by compound 6F was related to increased [Ca²⁺]_i in HL-60 cells, and DNA fragment induction was possibly catalyzed by Ca²⁺ - independent DNase.     

Changes of K+ channels of outer hair cells in guinea pig cochlea with streptomycin ototoxicity

AIM:To study the changes of K⁺ channels of outer hair cells in guinea pig cochlea with streptomycin ototoxicity. METHODS:Auditory brainstem responses (ABR) and whole-cell patch clamp techniques were used.RESULTS:(1) The body weight of guinea pigs with streptomycin ototoxicity decreased significantly; (2) The ABR threshold markedly increased in streptomycin group (Ⅱ,Ⅲ);(3)The number of dissociated outer hair cells of guinea pigs (Ⅱ,Ⅲ) was lower than that of control (Ⅰ); (4) Streptomycin decreased the Ca²⁺-sensitive K⁺ currents and delayed outward K⁺ currents distinctly; (5) There was no significant difference of K⁺ currents between Ⅰ and Ⅱ/Ⅲ. CONCLUSION:These results suggest that the inhibition of K⁺ channels is the basis of streptomycin ototoxicity, but not the direct reason for cell death.     

Nuclear calcium oscillation in cultured neonatal rat myocytes

AIM: To determine whether nuclear Ca²⁺ is independently regulated from the cytosolic Ca²⁺ and nuclear Ca²⁺ oscillation induced by many modulating factors in cultured rat neonatal myocytes and its mechanism. METHODS: Rat neonatal cardiac myocytes were cultured, and fluo-4/AM was loaded as calcium probe. The changes of cytosolic and nuclear Ca²⁺ were observed by confocal laser microscopy. RESULTS: Calcium fluorescent intensity oscillated slightly in myocardiocytes and the average intensity was much higher in the nucleus than that in the cytosole. Ca²⁺ oscillation in nucleus and cytosole induced by norepinephrine, isoproperenol, ATP were completely blocked by Ca²⁺-ATPase antagonist thapsigargin (10^-6 mol/L),L-type Ca²⁺ channel blocker verapermil (500 μmol/L) and KCl (20 mmol/L). Ca²⁺-ATPase antagonist thapsigargin completely blocked the propagation of Ca²⁺ waves and simutaneouly induced a temporary Ca²⁺ increase followed by a magnificient drop and loss of response to norepinephrine. CONCLUSIONS: The results suggested that generation and maintenance of calcium oscillation both in cytosole and nucleus depended on extracellular Ca²⁺ influx, membrane potential, Ca²⁺ release and uptake of cytosolic and nuclear calcium stores. The difference between cytosolic calcium and nuclear calcium indicated that calcium regulating system relatively independent of cytosole may exist in nucleus.     

Role of K+ channels in hypoxic pulmonary vasoconstriction in normal and chronic hypoxic rats

AIM:To investigate the role of K⁺ channels in the decreased hypoxic pulmonary vasoconstriction(HPV) in chronic hypoxic rats. METHODS:Blockers of three kinds of K⁺ channels, 4-AP(voltage dependent K⁺ channel blocker), TEA(Ca²⁺ activated K⁺ channel blocker), GLIB(ATP sensitive K⁺ channel blocker) were used in isolated perfused rat lungs to detect the role of K⁺ channels in HPV. RESULTS:In normal rats, 4-AP and TEA, but not GLIB, both elicited a significant increase in pulmonary artery baseline pressure, and also potentiated the acute hypoxic pulmonary vasoconstriction. In chronic hypoxic rats, the HPV is significantly decreased, while 4-AP, TEA, GLIB all elicited a significant but smaller increase in pulmonary artery baseline pressure. Additionally, all these three blockers potentiated the HPV stronger in chronic hypoxic rats than in control rats. CONCLUSION:The opening of Kv, K_Ca, K_ATP might modulate the hypoxic pulmonary vasoconstriction in isolated rat lungs, and the increase in this modulation by potassium channel in chronic hypoxic rats might play a role in its decrease in HPV.     

Roles of ERKs and intracellular free calcium in cardiomyocyte hypertrophic response induced by endothelin-1

AIM: To study the roles and mechanisms of ERKs and intracellular free calcium in cardiomyocyte hypertrophic response induced by endothelin-1(ET-1). METHODS: (1) Neonatal rat cardiomyocyte hypertrophic response was assayed by measuring cell surface area and protein content; (2) ERKs activity was determined by Whatman Paper Filter method; (3) Intracellular free calcium concentration ([Ca²⁺]i) was measured using Fura-2/AM as a fluorescent indicator. RESULTS:(1)ET-1 could increase total protein production,surface area,ERKs activity and[Ca²⁺]i in cultured cardiomyocyte in dose-dependent manner at concentrations ranging from 10^-9to 10^-7mol/L.And this effect could be abolished by BQ123,an antagonist of ETA receptor,partly inhibited by PTX,but not by BQ788,an antagonist of ETB receptor.(2)The activation of ERKs and the increase of[Ca²⁺]i induced by ET-1 were obviously in hibited by PD98059,a selective ERKs kinase inhibitor,and nifedipine,a calcium channel blocker,respectively.Both antagonists partialy inhibited ET-1-stimulated cardiomyocyte hypertrophic response.(3)Staurosporine,a selective PKC inhibitor,could inhibit ET-1-stimulated cardiomyocyte hypertrophic response and increase of[Ca²⁺]i,but not af ect the activation of ERKs.CONCLUSION: Cardiomyocyte hypertrophic response induced by ET-1 is mediated by ET_A receptor coupled to PTX-sensitive G-protein, which involves at least two signalling pathways: PKC-mediated increase of [Ca²⁺]i , and PKC-independent activation of ERKs.     

Interleukin-2 altered the frequency -dependent relationship of intracellular calcium in rat ventricular myocytes

AIM:We examined the effect of interleukin-2 (IL-2) on calcium handling of rat cardiomyocytes. METHODS:The effects of steady state and transient changes in stimulus frequency on the intracellular calcium transient were investigated in the isolated ventricular myocytes with spectrofluorometry technique. RESULTS: Under the steady state (0.2 Hz), IL-2 at 2×10⁵U/L decreased the peak [Ca²⁺] i and amplitude of the [Ca²⁺]i transient, increased the diastolic calcium level, and prolonged the decay of the calcium transient. At 1.25 mmol/L of extracellular [Ca²⁺], when increasing the stimulus frequency from 0.2 to 1.0 Hz, diastolic calcium level and peak [Ca²⁺] i as well as the amplitude of the transient were increased. The positive frequency relationship was blunted in the IL-2-treated myocytes and this was not normalized by increasing extracellular [Ca²⁺] to 2.5 mmol/L. The caffeine induced Ca²⁺ release was increased with increase in stimulus frequency. IL-2 inhibited the frequency relationship of caffeine induced Ca²⁺ release. The restitution was not different between control and IL-2 groups at the 1.25 mmol/L of extracellular [Ca²⁺], which was slowed in IL-2-treated myocytes when the extracellular [Ca²⁺] was increased to 2.5 mmol/L. CONCLUSIONS:It is concluded that the blunted frequency response of IL-2-treated myocytes was resulted from the decrease in SR Ca²⁺ release, which was related to depression of SR function. Despite the evidence of depressed SR Ca²⁺ uptake, the restitution of calcium transient at 1.25 mmol/L of extracellular remains unchanged, which maybe due to the increase in the Na⁺/Ca²⁺ exchanger activity.     

Relaxation effect of isoliensinine on isolated mouse airway smooth muscle

AIM: To study the relaxation effect of isoliensinine on high K⁺-induced isolated mouse airway smooth muscle (ASM) and the underlying mechanism. METHODS: The muscle tension transducer was used to detect the effects of isoliensinine on high K⁺-induced precontraction and Ca²⁺ influx in ASM. The technique of patch-clamp and calcium imaging system were respectively used to examine the effects of isoliensinine on LVDCC currents and[Ca²⁺]_i of the ASM cells (ASMCs). RESULTS: Isoliensinine significantly relaxed precontracted ASM induced by high K⁺ in a concentration-dependent manner. The maximum relaxation ratio was(95.3±3.9)% by isoliensinine at 100 μmol/L. In addition, LVDCC currents were measured using the whole-cell patch-clamp technique, which were abolished by isoliensinine. High K⁺-induced 340/380 nm fluorescence ratio of Fura-2 was 0.63±0.10 in ASMCs, while it decreased to 0.36±0.05 after the addition of isoliensinine (P<0.01). When isoliensinine was added at the peak point of[Ca²⁺]_i, the ratio rapidly decreased from 0.74±0.02 to 0.42±0.05 (P<0.01). Moreover, isoliensinine inhibited high K⁺-induced Ca²⁺ influx-mediated contraction of ASM. CONCLUSION: Isoliensinine inhibits LVDCC currents, terminates Ca²⁺ influx and reduces[Ca²⁺]_i, eventually resulting in relaxation of the ASM, indicating isoliensinine might be a potential bronchodilator.     

Interaction between STIM1 and Orai1 in calcium-sensing receptor-mediated calcium influx and nitric oxide generation

AIM: To investigate the interaction of Ca²⁺-sensing proteins, stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (Orai1), in Ca²⁺-sensing receptor (CaSR)-mediated extracellular Ca²⁺ influx and production of nitric oxide (NO). METHODS: Human umbilical vein endothlial cells (HUVECs) were incubated with CaSR agonist spermine [activating store-operated calcium channels (SOC) and receptor-operated calcium channels (ROC)] alone or combined with CaSR negative allosteric modulator Calhex 231+ROC analogue TPA (activating ROC, blocking SOC), protein kinase C (PKC) inhibitor Ro 31-8220, or PKCα/β1 selective inhibitor Go 6976 (activate SOC, blocking ROC). The protein expression of STIM1 and Orai1 was determined by the method of immunofluorescence. The interaction between STIM1 and Orai1 was examined by co-immunoprecipitation. The second to third passages of HUVECs were divided into STIM1 and Orai1 short hairpin RNA group (shSTIM1+shOrai1 group), vehicle-STIM1+vehicle-Orai1 group and control group, and then incubated with the 4 different treatments above. The intracellular Ca²⁺ concentration ([Ca²⁺]_i) was detected using the fluorescent Ca²⁺ indicator Fura-2/AM. The production of NO was also determined by DAF-FM DA fluorescent probe. RESULTS: The protein expression of STIM1 and Orai1 was located in the cytoplasm. Compared with control group, the localization of STIM1 and Orai1 in the cytoplasm was reduced after the HUVECs were incubated with Calhex 231+TPA, Ro 31-8220 or Go 6976, and the interaction of STIM1 and Orai1 was decreased significantly. The [Ca²⁺]_i and the net NO fluorescence intensity in shSTIM1+shOrai1 group were significantly reduced after the 4 different treatments (P<0.05). CONCLUSION: STIM1 and Orai1 are components of SOC and ROC in store-and receptor-operated Ca²⁺ entry and NO generation.     

Role of ClC-3 chloride channel in inhibition of nasopharyngeal carcinoma cell cycle by metformin

AIM: To study the roles of ClC-3 chloride channel in the inhibition of nasopharyngeal carcinoma cell cycle by metformin. METHODS: The CNE-2Z cells were treated with metformin at different concentrations. The viability of CNE-2Z cells was measured by CCK-8 assay. The cell cycle distribution was detected by flow cytometry. The protein expression of ClC-3 was determined by Western blot. The Cl^- currents was record by the patch-clamp technique. In addition, the cell cycle distribution was analyzed in the nasopharyngeal carcinoma CNE-2Z cells which over-expressed ClC-3 by pEZ-M03-ClC-3 plasmid transfection. RESULTS: Metformin inhibited the viability of CNE-2Z cells at 5, 10 and 20 mmol/L. Metformin at 10 mmol/L prevented the activation of chloride currents induced by hypotonicity, inhibited the protein expression of ClC-3 chloride channel and arrested the nasopharyngeal carcinoma CNE-2Z cells at G₀/G₁ phases. ClC-3 chloride channel protein over-expression reversed the effect of metformin on the cell cycle distribution of CNE-2Z cells. CONCLUSION: Metformin inhibits the CNE-2Z cell cycle, which may be related to the inhibition of ClC-3 chloride channel function and protein expression.     

Effect of NCA on excitation-contraction coupling of cardiac muscle from phospholamban knockout mice

AIM: Nitroxyl(HNO) increases myofilament Ca²⁺ responsiveness relative to increases in intracellular Ca²⁺ in cardiac muscle. In this study, we further investigated this effect of HNO on trabecular muscles from phospholamban knockout(PLB-KO) and wide-type(WT) mice using a novel HNO donor, 1-nitrosocyclohexyl acetate(NCA). METHODS: Trabecular muscles were dissected from the right ventricles of the rat hearts and mounted between a force transducer and a motor arm. The muscles were superfused with K-H solution(pH 7.4) at room temperature. Fura-2 was loaded into the trabecular muscles via electrophoresis. The length of the sarcomere was set to 2.2~2.3μm. During steady-state activations, the maximal Ca²⁺-activated force and Ca²⁺ required for 50% activation were measured. RESULTS: The intracellular Ca²⁺ transients and force of the PLB-KO muscles at baseline were higher than those of the WT muscles and exhibited a negative force-frequency relationship(FFR). NCA(2.5μmol/L) increased systolic force in both PLB-KO group and WT group at any given[Ca²⁺]_o. However, there was more dramatic increase in the force development due to moderate increases in the intracellular Ca²⁺ transients in the WT muscles when external Ca²⁺ increased from 1.5 to 4.5 mmol/L under NCA. NCA did not affect the negative FFR in PLB-KO muscle. Steady-state force-Ca²⁺ relations obtained from skinned muscles were not different between the 2 groups, while NCA increased Ca²⁺ responsiveness in skinned muscles from both PLB-KO and WT mice.CONCLUSION: HNO increases force development in both PLB-KO and WT muscles as a result of increases in myofilament Ca²⁺ responsiveness. The increased intracellular Ca²⁺ transients are accompanied by greater force development in WT mice, suggesting that HNO improves Ca²⁺ activation and establishes HNO as a positive inotropic agent with novel mechanisms.     

Importance of mitochondrial calcium uniporter in high glucose-induced cardiac myocyte apoptosis

AIM: To investigate the role of mitochondrial calcium uniporter (MCU) in high glucose(HG)-induced apoptosis of cardiac myocytes. METHODS: Cardiac myocytes were exposed to normal glucose (5.5 mmol/L glucose+ 19.5 mmol/L mannitol), HG (25 mmol/L glucose), or HG combined with 5 μmol/L spermine for 72 h. Mitochondrial free Ca²⁺ concentration ([Ca²⁺]_m), MCU at mRNA and protein levels, pyruvate dehydrogenase (PDH) activity, mitochondrial membrane potential (Δψ_m), the levels of ATP and reactive oxygen species (ROS), and apoptosis were determined. RESULTS: The [Ca²⁺]_m, the mRNA and protein levels of MCU, PDH activity, ATP levels, and Δψ_m were reduced (P<0.05), while ROS content and the protein levels of caspase-9 and caspase-3 were increased in HG group (P<0.05). Adding 5 μmol/L spermine returned these parameters toward control levels (P<0.05). Moreover, apoptosis was reduced by adding spermine and HG treatment (P<0.05). CONCLUSION: HG-induced cardiac myocyte apoptosis may be associated with the decreased MCU expression and activity, abnormal mitochondrial Ca²⁺ handling, deviant mitochon-drial respiratory chain, and mitochondrial dysfunction.     

The alteration of extracellular sodium and potassium ionic activities of hippocampal neurons in epileptiform rats kindled by coriaria lactone

AIM and METHODS: The sodium ion Na⁺ and potassium ion K⁺ selective microelectrodes were used to measure changes of ionic activity of extracellular sodium and potassium( [Na⁺]_o, [K⁺]_o) in hippocampus and hippocampal slice during epieptic seizure induced by intrahippocampal microinjection of coriaria lactone(CL) in rats and perfusing hippocampal slice with CL. RESULTS:30 s, 1min and 2min after injection of CL into hippocampus, the [Na⁺]_o decreased 27.7 mmol/L, 50.3 mmol/L, 57.8 mmol/L respectively and the [K⁺]_o increased 2.3 mmol/L, 2.4 mmol/L, 2.9 mmol/L respectively compared with control values(P＜0.01). The [K⁺]_o returned to the control level 3min after local application of CL, but the[Na⁺] _o was still lower than that of control group(P＜0.01). The [Na⁺]_o and the [K⁺]_o were measured also in hippocampal silces and results are similar to those of experiments in vivo. CONCLUSION: The influx of Na⁺and the flux of K⁺occurred during epileptiform discharges of hippocampal neurons induced by administration of CL.     

Trx-1 over-expression attenuates oxidative stress injury in MPP+ -induced PC12 cells by regulating NF-κB signaling pathway

AIM:To investigate the inhibitory effect of thioredoxin 1 (Trx-1) over-expression on oxidative stress injury in 1-methyl-4-phenylpyridinium (MPP⁺)-induced rat pheochromocytoma PC12 cells by regulating NF-κB signaling pathway.METHODS:The PC12 cells were damaged by treatment with MPP⁺ at 1, 3 and 5 mmol/L, and the optimal concentration of 3 mmol/L was selected. The cell viability was measured by MTT assay. The oxidative stress indexes lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in the cell culture supernatant were detected, and the protein expression of Trx-1 was determined by Western blot. Lentiviral infection with Ad-Trx-1-GFP sequence was used to establish a model of MPP⁺-treated PC12 cells with Trx-1 over-expression. The effects of Trx-1 over-expression on the cell viability, oxidative stress responses and NF-κB signaling pathway were determined by MTT assay, commercial kits and Western blot. The effects of phorbol 12-myristate 13-acetate (PMA), an activator of NF-κB signaling pathway, on the viability and oxidative stress of PC12 cells were observed. The NF-κB signaling pathway inhibitor pyrrolidine dithiocarbamate (PDTC) was used in MPP⁺-treated PC12 cells with Trx-1 over-expression, and the cell viability and oxidative stress responses were measured. RESULTS:The viability of PC12 cells, SOD activity in the supernatant and protein expression of Trx-1 were decreased, while LDH activity and MDA content in the supernatant were increased significantly by treatment with MPP⁺ at 1, 3 and 5 mmol/L. The effect of MPP⁺ at 3 mmol/L and 5 mmol/L was significantly greater than that at 1 mmol/L (P<0.05), and no significant difference between 3 mmol/L and 5 mmol/L was observed (P>0.05). The inhibitory effect of MPP⁺ on the viability of PC12 cells, and the oxidative stress injury and activation of NF-κB signaling pathway induced by MPP⁺ were significantly attenuated by over-expression of Trx-1. The inhibitory effect of MPP⁺ on the viability of PC12 cells and the oxidative stress injury induced by MPP⁺ were promoted by the activation of NF-κB signaling pathway, while the protective effects of Trx-1 over-expression on the MPP⁺-treated PC12 cells were enhanced by the inhibition of NF-κB signaling pathway. CONCLUSION:Over-expression of Trx-1 protects MPP⁺-treated PC12 cells from oxidative stress injury by regulating NF-κB signaling pathway.     

Intracellular calcium alterations induced by endotoxin and IL-1β in rabbit hypothalamic neurons

AIM: To investigate intracellular free calcium ( [Ca²⁺]i ) alterations in hypothalamus of febrile rabbits induced by endotoxin (ET), and compare with the effect of ET and IL-1β on i in hypothalamic neurocytes from normothermia rabbits. METHOD: The concentration of [Ca²⁺]i was determined by using spectrofluorometer and fluorescent Ca²⁺ probe fura-2 /Am. RESULTS: 1. A minute dose of ET (2 ng/mL) induced a significant rise in [Ca²⁺]i in hypothalamic neurocytes from normothermia rabbits. The rise in [Ca²⁺]i in hypothalamic neurocytes from febrile rabbits induced by intravenous injection of ET was also observed. 2. In hypothalamic neurocytes from normotheria rabbits, IL-1β failed to affect [Ca²⁺]i at concentrations of 100, 500, 1 000ng/mL, respectively. CONCLUSION:The action site of low concentration of calcium that plays a regulatory role during fever seems unlikely to be in cytosolic compartment of hypothalamic neurons. The change of [Ca²⁺]i in hypothalamic neurocytes by ET can not be considered the direct effect of IL-1β.     

Effects of magnesium on apoptosis and expression of Foxp3 in peripheral CD4+CD25+ regulatory T cells from asthma patients

AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4⁺CD25⁺ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4⁺CD25⁺ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4⁺CD25⁺T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4⁺CD25⁺ T cells in CD4⁺T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4⁺CD25⁺ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4⁺CD25⁺ regulatory T cells.     

High glucose induces H9c2 cardiomyocyte hypertrophy through Ca2+-CaN-NFAT3 signaling pathway

AIM: To study the morphological changes of cardiac H9c2 cells during the developmental process of fetal rat. METHODS: Embryonic rat heart-derived H9c2 cells were maintained in DMEM supplemented with 10% fetal bovine serum. The H9c2 cells were plated at a density of 6000 cells/cm and divided into 5 groups: H9c2 cells were treated with 5 mmol/L glucose, 25 mmol/L glucose, 50 mmol/L glucose, Norvasc (25 nmol/L)+25 mmol/L glucose, or Norvasc (25 nmol/L)+50 mmol/L glucose for 48 h. The morphology of H9c2 cells was observed. The cell surface area was measured by Image-Pro Plus 6.1 software. Fluorescence spectrophotometry was used to detect the concentration of intracellular calcium ion ([Ca²⁺]_i)in the cardiomyocytes. The concentration of CaN in the cell was measured by ELISA. The mRNA expression of CaNAβ, NFAT3 and β-MHC in the cells was detected by real-time PCR. The protein levels of CaNAβ, NFAT3 and β-MHC in cultural H9c2 cells were detected by Western blot. RESULTS: The mean area of the cells, the mean fluorescence value of [Ca²⁺]_i and the concentration of CaN in 25 mmol/L glucose group were higher than those in 5 mmol/L glucose group, and those were lower than those in 50 mmol/L glucose group. After treated with Norvasc, those results decreased significantly. The expression of CaNAβ, NFAT3 and β-MHC at mRNA and protein levels in 25 mmol/L glucose group was higher than those in 5 mmol/L glucose group, but was lower than those in 50 mmol/L glucose group. The expression of CaNAβ, NFAT3 and β-MHC at mRNA and protein levels decreased significantly in Norvasc treatment group. CONCLUSION: Ca²⁺-CaN-NFAT3 signaling pathway is perhaps involved in high glucose-induced H9c2 cardiomyocyte hypertrophy.