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Objective To understand the differences in killing ability and mechanism of human mononuclear macrophages and neutrophils against Leptospira interrogans.Methods Human THP-1 and HL-60 cell lines were respectively pretreated with PMA (phorbol 12-myristate 13-acetate) and ATRA (all-trans retinoic acid) to induce their differentiation into macrophages and neutrophils.Confocal microscopy was used to detect the changes in total ROS (reactive oxygen species) and NO (nitric oxide) levels as well as free Ca2+ concentration ([Ca2+]i) in THP-1 monocyte-derived macrophages and HL-60 cell-derived neutrophils after infection with Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai.Fluorospectrophotometry was applied to analyze the differences in killing ability between THP-1 monocyte-derived macrophages and HL-60 cell-derived neutrophils against intracellular leptospires before and after treatment with total ROS and NO inhibitors and intracellular free Ca2+ chelator.Results The total ROS and NO levels and [Ca2+]i in THP-1 monocyte-derived macrophages and HL-60 cell-derived neutrophils were significantly increased after infection with the spirochete (P<0.05).Moreover,the total ROS and NO levels and [Ca2+]i in the former were significantly higher than those in the latter (P<0.05).The THP-1 monocyte-derived macrophages had stronger killing ability against intracellular leptospires than the HL-60 cell-derived neutrophils (P<0.05).Inhibiting total intracellular ROS,NO or free Ca2+ could result in decreased killing ability of THP-1 monocyte-derived macrophages against intracellular leptospires,but not affect the killing ability of HL-60 cell-derived neutrophils.Conclusion Mononuclear macrophages rather than neutrophils act as the main phagocytes eliminating Leptospira interrogans.High levels of total intracellular ROS,NO and free Ca2+ are closely associated with the ability of mononuclear macrophages to kill Leptospira interrogans.
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To investigate the relationship between intracellular free Ca2+ concentration ([Ca2+]i)and calcium-activated chloride (Clca) channels of pulmonary artery smooth muscle cells (PASMCs) in rats under acute and chronic hypoxic conditions, acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca2+indicator Fura-2/AM was used to observe [Ca2+]i of rat PASMCs under normal and chronic hypoxic condition. The effect of Clca channels on PASMCs proliferation was assessed by MTT assay.The Clca channel blockersniflumic acid (NFA) and indaryloxyacetic acid (IAA-94) exerted inhibitory effects on acute hypoxia-evoked contractions in the pulmonary artery. Under chronic hypoxic condition, [Ca2+ ]i was increased. Under normoxic condition, [Ca2+]i was (123.63±18.98) nmol/L, and in hypoxic condition, [Ca2+]i was (281. 75±16.48) nmol/L (P＜0.01). Under normoxic condition, [Ca2+]i showed no significant change and no effect on Clca channels was observed (P＞0. 05). Chronic hypoxia increased [Ca2+]i which opened Clca channels. The NFA and IAA-94blocked the channels and decreased [Ca2+]i from (281. 75±16.48) nmol/L to (117.66±15.36)nmol/L (P＜0.01). MTT assay showed that under chronic hypoxic condition NFA and IAA-94 decreased the value of absorbency (A value) from 0. 459±0. 058 to 0. 224±0. 025 (P＜0.01).Hypoxia increased [Ca2+]i which opened Clca channels and had a positive-feedback in [Ca2+]i. Thismay play an important role in hypoxic pulmonary hypertension. Under chronic hypoxic condition,Clca channel may play a part in the regulation of proliferation of PASMCs.
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Objective To explore the double-labeling method of monitoring the GHRP regulatory function on [Ca2+]i and NO in cardiomyocytes of rats on real time under LSCM.Methods The reformed constant-flow Langendorff system and enzyme-dissociated was used to isolate cardiomyocytes.[Ca2+]i and NO in the cardiomyocytes of SD rats were double-labeled by their molecular probe Rhod-2/AM and DAF-FM/DA,respectively to monitor the regulatory function of GHRP on [Ca2+]i and NO on real time by LSCM.Results Ca2+ signal showed a red fluorescence and NO showed a green fluorescence while the overlapping of the two signals showed a yellow-green fluorescence by this system,and the similar effect presents in both double-labeled state and the single labeled one:GHRP induced a transient[Ca2+]i increase then followed by a plateau phase while there was not significant change in NO signal system after GHRP stimulation under the LSCM in the cardiomyocytes of rats.Conclusions After having established the double-labeling method we monitored the GHRP regulatory function on [Ca2+]i and NO on real time in cardiomyocytes of rats under LSCM causing the [Ca2+]i biphasic increase while no significant change in NO signal system.
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AIM: To investigate the protective effects and mechanisms of iptakalim hydrochloride(Ipt)on H_2O_2 induced neurotoxity. METHODS: Neurotoxity injury was induced by H_2O_2 in PC12 cells. The cell viability was tested by MTT assay. The glutamate released from PC12 cells was measured by HPLC combined with fluorescent detector analysis. Changes in the intracellular free Ca 2+ concentration ([Ca 2+ ]_i) were determined in fluo-3 AM loaded PC12 cells. RESULTS: Ipt (1, 10 and 100 ?mol?L -1 ) markedly mitigated H_2O_2-induced neurotoxity, 10 ?mol?L -1 Ipt inhibited the release of glutamate and the increase of [Ca 2+ ]_i induced by H_2O_2 .The protective effects was incompletely blocked by 5-HD which is a mitochondrial K_ ATP channels antagnist. CONCLUSION: Ipt provides neuroprotective effects on H_2O_2 induced cytoxixity in cultured PC12 cells and the protective effects may be partially related with mitochondrial KATP channels.
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Objective:To observe the effect of Xinmailong injection (XI) on free calcium ( Ca2+) content in rat myocardial cells. Methods:Free Ca2+content in rats treated with various dosages of XI were measured by fluorospectrophotometry. Results:Xinmailong injection significantly increased free Ca2+content and the content was 169.18?11.64, 233.26?10.69 and 164.25?10.34 nmol?L-1 in 0.19 g?L-1 , 0.38 g?L-1 and 0.76 g?L-1 XI group respectively, the differences being significant as compared with 120.64?3.02 nmol?L-1 in the control group (P
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OBJECTIVE:To investigate the effects of artesunate(ART) on free Ca2+ concentration([Ca2+]i) in human prostatic cancer PC-3 cells.METHODS:PC-3 cells were treated with ART and labeled using Ca2+ fluorescent probe Fluo-3/AM,and the change of[Ca2+]i was evaluated by confocal laser scanning microscopy.RESULTS:After PC-3 cells were exposed to artesunate,the intracellular [Ca2+]i concentration increased significantly at 0～0.5 h,maintained at a high level at 0.5～1 h,but decreased to the level of the initial at 4～24 h. CONCLUSION:ART can induce the marked sustained increase of[Ca2+]i and [Ca2+]i might play an important role in ART-induced apoptosis of human PC-3 cells.
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AIM To study the effects of A? 25～35 and Apo E4 on neuronal intracellular free Ca 2+ ([Ca 2+ ] i). METHODS Hippocampal and cortical neurons suspension of newborn(0～3 days) SD rats was produced. After incubated with fura 2/AM,the neurons suspension was divided into four groups: control, A? 25～35 , Apo E4, A? 25～35 +Apo E4. Each groups [Ca 2+ ] i was measured using a RF 5000 dual wavelength spectrofluorometer after incubated with double distilled water, A? 25～35 , Apo E4, A? 25～35 +Apo E4 for 3 min, respectively. The neurons outocorrelation function(ACF) of the scattering light intersity was analyzed by the microscope quasi elastic light scattering(MQLS) technique The frequency shift line width by ACF. The ? can sympolize the cell menbrane flilidity. RESULTS Both A? 25～35 and Apo E4 could significantly enhance hippocampal and cortical neurons rest [Ca 2+ ] i, furthermore, the effect of 5 ?mol?L -1 A? 25～35 was higher than the effect of 1 ?mol?L -1 A? 25～35 ( P