Regulation of LPS-induced elevation of Ca2+ intracellular level of alveolarmacrophages in chronic bronchitis by Angelica Sinensis and nifedipine

AIM: To explore regulation of lipopolysaccharide (LPS)-induced elevation of Ca²⁺ intracellular level in alveolar macrophages(AMs) from patients with chronic bronchitis by Angelica Sinensis and nifedipine.METHODS:AMs was obtained from 7 patients with chronic bronchitis and 6 normal controls by bronchoalveolar lavage and intracellular Calevel was detected after adding Angelica Sinensis, nifedipine or LPS to the supernatant of AMs loaded by Fura-2. RESULTS: In contrast with normal control group (99.65±32.21 nmol/L), intracellular Ca²⁺ level in AMs from chronic bronchitis group (189.47±23.69 nmol/L) was increased significantly in the absence of extracellular Ca²⁺ but not 1 mmol/L. Intracellular Ca²⁺ level in AMs from chronic bronchitis group were significantly increased by adding 10 μg/mL LPS to the supernatant of AMs. LPS-induced elevation of intracellular Ca²⁺ level in AMs from chronic bronchitis group was completely inhibited by Angelica Sinensis or nifedipine.CONCLUSION: Both Anelica Sinensis and nifedipine may inhibit activation of AMs from patients with chronic bronchitis by reducing LPS-induced elevation of intracellular Ca²⁺ level in AMs, suggested that these two medicines may inhibit non-specific inflammation of airways in chronic bronchitis.     

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.     

Changes of intracellular Ca2+ in living brain slices during focal cerebral ischemia/reperfusion

AIM: The purpose of the present study was to detect intracellular Ca²⁺changes in living brain slices during focal cerebral ischemia/reperfusion (I/R) and reveal the role of intracellular Ca²⁺in the cerebral I/R injury. METHODS: The model of focal cerebral I/R was established in rats by reversible inserting a nylon thread, and dynamic change of intracellular Ca²⁺in brain slices was determined using laser confocal imaging system. RESULTS: ① Ca²⁺gradually enhanced with increase in ischemic time in cortex and striatum. ②At1h ischemia/10min reperfusion, Ca²⁺increased significantly in striatum, but Ca²⁺decreased at 3 h reperfusion compared with10min reperfusion. ③ Ca²⁺markedly enhanced at 6 h ischemia compared with1h ischemia, and after 3 h reperfusion Ca²⁺decreased, but was still higher than that in sham-operation group. ④The striatum is more sensitive than cortex to ischemia/reperfusion. CONCLUSION: Ca²⁺overload in the area of cortex and striatum may play an important role in cerebral ischemia/reperfusion injury in rats.     

Salidroside increases release of intracellular free Ca2+ from sarcoplasmic reticulum in cultured rat cardiomyocytes

AIM: To observe the effects of salidroside on intracellular free Ca²⁺ concentration in cultured rat cardiomyocytes. METHODS: Primarily cultured cardiomyocytes of neonatal rats were divided into control group, different concentrations of salidroside groups and verapamil pretreatment+different concentrations of salidroside groups. The fluorescent intensity of intercellular free calcium concentration ([Ca²⁺]_i) in cultured cardiomyocytes of newborn rats loaded with fluo-3/AM(5 μmol/L) was measured by laser scanning confocal microscopy. RESULTS: Salidroside at concentrations of 15 mg/L, 30 mg/L and 60 mg/L elevated [Ca²⁺]_i in cultured rat cardiomyocytes with the peak values of 574.08±4.65, 591.86±3.64 and 618.66±4.27, respectively (all P<0.01), indicating that the effect of salidroside on the level of [Ca²⁺]_i was dose-dependent. In the presence of verapamil in D-Hanks solution, salidroside also elevated the fluorescent intensity of [Ca²⁺]_i in cardiomyocytes from 357.74±3.13, 387.17±2.37 and 391.43±1.34 to 480.86±3.98, 496.70±3.08 and 522.18±3.19, respectively (all P<0.01). CONCLUSION: Salidroside increases the release of [Ca²⁺]_i from sarcoplasmic reticulum in cultured rat cardiomyocytes.     

Modulation of K+ channels by Ca2+ and pH in regulatory volume decrease

It has been widely appreciated that animal cells rely on the mechanism of regulatory volume decrease(RVD) after swell in a hypotonic environment. Activation of K channels is crucial in the process of self-protection. There are a characterized increase in cytoplasmic Ca and a decrease in pH in the process of RVD in many cell types. Ca entry via transient receptor potential(TRP) channel is crucial for the cytoplasmic Ca increase, which in turn induces the decrease in pH.The increase in cytoplasmic Ca and decrease in pH activate or inhibit the activity of K channel, respectively. In this review, the regulatory network at cellular level between cytoplasmic Ca and pH, and the modulation of K channels by Ca and pH in the process of RVD are discussed.     

Influences of Ca2＋ antagonist on the mRNA levels of L-type Ca2＋ channel and Na＋-Ca2＋ exchanger in the atria of hypertensive rats

AIM: To investigate the mRNA changes of L-type Ca²⁺channel α_1Csubunit (CaL-α_1C) and Na⁺-Ca²⁺ exchanger (NCX) in the atria of renovascular hypertensive rats. METHODS: Two-kidney one-clip Goldblatt hypertensive Sprague-Dawley rats were divided into three groups 1 week after operation, HD group was treated with 250 mg/d diltiazem, LD group was treated with 50 mg/d diltiazem, control group (C group) was treated with vehicle. After 4 weeks treatment, semiquantitative RT-PCR was used to estimate the mRNA changes of CaL-α_1Cand NCX, and GAPDH was used as internal control. RESULTS: Systolic blood pressure in LD group was comparable with C group, and that in HD group was decreased to normal level after diltiazem treatment. In C group, CaL-α_1C mRNA level were 2.5, 2.4 and 2.1 times of S, HD and LD group, and NCX mRNA level were 1.9, 1.6 and 2.1 times of S, HD and LD group. There were no significant difference in the mRNA level of CaL-α_1Cand NCX among S, HD and LD group. CONCLUSION: The mRNA levels of CaL-α_1Cand NCX are upregulated in the atria of hypertensive rats. Ca²⁺ antagonist inhibites their upregulation independent of blood pressure.     

Quercetin inhibits endothelin-1-induced T-type Ca2+ channel expression in human umbilical arterial smooth muscle cells

AIM: To investigate the effect of quercetin on endothelin-1-induced T-type calcium channel(TCC) expression in primary cultured human umbilical arterial smooth muscle cells for exploring the protective role of quercetin in cardiovascular system. METHODS: Human umbilical arterial smooth muscle cells were verified by immunocytochemistry. The cells in 2-3 passages were used and randomly divided into control group, quercetin alone group, model group and experimental group. The cells in control group were cultured without any drugs for 24 h. The cells in quercetin alone group were cultured with 80 μmol/L quercetin for 24 h. The cells in model group were cultured with ET-1 at the concentration of 100 nmol/L for 24 h. The cells in experimental groups were pretreated with quercetin for 1 h, then coincubated with 100 nmol/L ET-1 for 24 h. The concentrations of quercetin used in this study were 20, 40and 80 μmol/L, respectively. The expression of α_1G, a TCC major subunit, was assayed at mRNA and protein levels by RT-PCR and Western blotting, respectively. The TCC currents(I_caT) were detected by the technique of whole-cell patch-clamp. RESULTS: Compared with control and experimental group, I_CaT density (P<0.01) and the expression of α_1G at mRNA (P<0.05) and protein (P<0.01) levels in model group were significantly increased. No significant difference in the results of quercetin alone group and control group was observed. CONCLUSION: The protective roles of quercetin in cardiovascular functions are related to the depressive effects of quercetin on ET-1-induced increase in both I_CaT density and the expression of α_1G at mRNA and protein levels in cultured human vascular smooth muscle cells.     

ATPase activities and Ca2+/calmodulin alterations in hippocampi of rats with PTSD-like behaviors

AIM: To explore the pathophysiological bases in the pathogenesis of the lasting emotional behavioral disorders following posttraumatic stress disorder(PTSD). METHODS: 240 male Wistar rats were divided randomly into 3 groups. Group SE(n =96) for rats with PTSD-like behavior by constant pulsating current of 100 μA with intratrain frequencies of 16 Hz, pulsating duration of 1 ms, train duration of 10 s and interstimulus interval of 7 min for 5 days with 8 times per day. Group CE(n =96) for control with electrode implanted in hippocampus without stimulation, and Group NC(n =48) for normal control. The activities of Na⁺-K⁺-ATPase and Ca²⁺ -ATPase, levels of intracellular calcium and free calmodulin(CaM), and the total CaM expression were detected in hippocampi of experimental rats. RESULTS: The activities of Na⁺-K⁺-ATPase and Ca²⁺ -ATPase in mitochondria of hippocampal cells in Group SE rats were significantly decreased at 48 h and 72 h after the last stimulation, respectively. The intracellular free calcium levels were increased, and the mean channel fluorescence of intracellular free CaM decreased remarkably at 72 h poststimulation, while the expression of total CaM was significantly elevated at 48 h after the last stimulation in hippocampi of Group SE rats. CONCLUSION: The lasting increased levels of intracellular free calcium and expression of Ca²⁺ -CaM in hippocampus, as well as the dysfunction of Na⁺-K⁺ pump and Ca²⁺ -ATPase in mitochondria may play important roles in the long-term neuropsychological sequelae in PTSD.     

Role of potassium channels in the regulation of intracellular free Ca2+ concentration of pulmonary artery smooth muscle cells in rats

AIM: To investigate the role of potassium channels in the regulation of intracellular free calcium concentration ( [Ca²⁺]_i) of pulmonary artery smooth muscle cells (PASMCs) in rats. METHODS: The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe [Ca²⁺]_i of rat PASMCs in normal and chronic hypoxic condition. The influences of potassium channels on PASMCs proliferation were assessed by MTT assay. RESULTS: 1. In normoxic condition, [Ca²⁺]_i was (156.91±8.60) nmol/L, and in hypoxic condition, [Ca²⁺]_i was (294.01±16.81) nmol/L. 2. In normoxic condition, the voltage-dependent K⁺-channel antagonist 4-aminopyridine (4AP), but not the Ca²⁺-activated K⁺-channel antagonist tetraethylammonium (TEA) and the ATP-sensitive K⁺-channel antagonist glibenclamide (Glib) increased [Ca²⁺]_i. 3. In hypoxic condition, 4AP and TEA caused the rise in [Ca²⁺]_i , but Glib had no effect on [Ca²⁺]_i. 4. MTT assay showed that 4AP increased the value of absorbing light degree (A value) in normoxic and hypoxic condition (0.582±0.062,0.873±0.043,respectively, P＜0.01), TEA increased A value only in hypoxic condition, and Glib had no effect on the proliferation of PASMCs. CONCLUSIONS: K_V plays an important role in the regulation of [Ca²⁺]_i and proliferation of PASMCs. K_Ca serves as distinct responsive roles in the regulation of proliferation of PASMCs in hypoxic condition. K_ATP has no effect on [Ca²⁺]_i and proliferation of PASMCs in normoxic and hypoxic conditions.     

LPS induced macrophage priming effect on O2- production and its signal mechanisms

AIM:To investigate the effect of lipopolysaccharide (LPS) priming on macrophage(MΦ).METHODS:Macrophage cell line RAW264.7 were pretreated with or without LPS for 1 h, then challenged with PMA, or LPS, muramyl dipeptide(MDP), Zymosan, formyl-methionyl-leucyl-phenylalanine(FMLP) for 1 h. O₂^- production in supernatants and intracellular free calcium([Ca²⁺]i) were measured, and changes in [Ca²⁺]i and LPS induced O₂^- production were compared.RESULTS:LPS pretreatment significantly increased O₂^- production in RAW264.7 cells challenged with the stimuli, and in a certain extent, both O₂^- production and increase of resting intracellular [Ca²⁺]i were dose- and time-dependent on LPS pretreatment.Furthermore, the peak [Ca²⁺]i was significantly higher in LPS pretreated groups than that of LPS unpretreated groups when challenged with PMA. Pretreatment with Ca²⁺ inophore A23187 mimicked the LPS priming effects on O₂^- production, but pretreatment with Ca²⁺ chelator BAPTA and EGTA blocked this priming effect.CONCLUSION:LPS-induced MΦ priming effect on O₂^- production is dependent on elevation of resting intracellular [Ca²⁺]i.     

Influence of the serum of Radix Ophiopogonison the apoptosis related gene expression and intracellular Ca2+ in vascular endothelial cells treated with LPS

AIM:To explore the molecular mechanism of Radix Ophioponis against vascular endothelial cell (VEC) apoptosis induced by LPS.METHODS:The apoptosis of human umbilical vascular endothelial cells(HUVEC) was induced by lipopolysaccharide(LPS). The influence of Radix Ophiopogonis on the expression of bcl-2 and intracellular Ca²⁺ was detected by flow cytometry and laser confocal microscope.RESULTS:The serum containing Radix Ophiopogonis suppressed the increase in bcl-2 expression and overloading of Ca²⁺ induced by LPS in HUVEC.CONCLUSION:The mechanism of Radix Ophiopogonis against HUVEC apotosis may be related with its regulatory effect on bcl-2 expression and remission of Ca²⁺ overloading.     

Antagonistic effects of cyproheptadine and anisodamine on [Ca2+]i elevation induced by TNFα in endothelial cell strains

AIM: To study the effects of cyproheptadine (Cyp) and anisodamine (Ani) on the changes of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) induced by tumor necrosis factor (TNFα) in single endothelial cells, and to explore the mechanisms of TNFα mediated shock and antishock actions of Cyp and Ani. METHODS: Human umbilical vein endothelial cell strains (ECV304) were seed in 35 mm tissue culture dish with 2 mL DMEM culture medium. The cultured cells were loaded by Fluo-3/AM. The spatial distribution and the dynamic changes of [Ca²⁺]_i in single endothelial cell was determined by laser scanning confocal microscopy (LSCM). RESULTS: [Ca²⁺]_i in single endothelial cell after stimulation of TNFα rapidly increased in a dose-dependent manner and approached the peak value within 60 seconds, afterwards, decreased and kept above the basal level. The confocal scanning image showed that [Ca²⁺]_i elevation was more obvious in nuclear than in cytoplasma, and decreased slowly. Cyp (3×10^-5, 6×10^-5 mol/L) and Ani (2×10^-5, 4×10^-5 mol·L^-1) markedly inhibited TNFα (1.2×10^-9 mol·L^-1)-induced [Ca²⁺]_i elevation. CONCLUSIONS: TNFα markedly induces elevation of [Ca²⁺]_i in single endothelial cell, it may be an important mechanism of TNFα-induced shock and tissue injury. Cyp and Ani obviously suppress TNFα-induced [Ca²⁺]_i elevation, which probably is one of the mechanisms of their antishock effects.     

Lipophagy inhibits foam cell formation by reducing cholesterol content

AIM To observe the changes of lipophagy during foam cells formation, and to determine the effect of lipophagy on the lipid content and cholesterol outflow of foam cells. METHODS Human THP-1 monocytes were induced by phorbol-12-myristate-13-acetate for 48 h to differentiate into macrophages, and then were incubated with 50 mg/L oxidized low-density lipoprotein （oxLDL） to form foam cells. Lipids in foam cell were stained by oil red O, and the lipid content was determined. The total cholesterol （TC） and free cholesterol （FC） levels in foam cells were measured by cholesterol testing kit. Cholesteryl ester （CE） and CE/TC ratio were calculated. The cholesterol efflux rate was detected by cholesterol efflux assay kit. The expression of autophagy-related proteins, including autophagy-related protein 5 （Atg5）, microtubule-associated protein 1 light chain 3 （LC3） and P62, were detected by Western blot. The colocalization of lipid droplets （LD） and LC3 was detected by immunofluorescence staining. The autophagy inducer rapamycin （Rap） or blocker 3-methyladenine （3MA） was used to intervene foam cells, and the expression of Atg5, LC3 and P62, the co-expression of LD and LC3, the cholesterol content and the cholesterol efflux rate were determined. RESULTS Formation of foam cells was observed at 24 h after stimulation with oxLDL at 50 mg/L, as indicated by intracellular CE/TC ratio exceeding 50%.Cholesterol efflux assay revealed that the cholesterol efflux rate increased within 24 h during foam cell formation but decreased after 48 h （P<0.05）. Western blot results displayed that the expression of Atg5 and LC3-II/LC3-I ratio were increased within 24 h of foam cell formation, but was deceased after 48 h （P<0.05）. The expression of P62 was decreased within 24 h but was increased at 48 h （P<0.05）. The colocalization of LD and LC3 was increased at 24 h but was decreased at 48 h after oxLDL stimulation. Treatment with Rap up-regulated the expression of Atg5 and LC3-II/LC3-I ratio, reduced the level of P62, increased the colocalization of LD and LC3, promoted the cholesterol efflux, anf reduced cholesterol content in foam cells （P<0.05）. On the contrary, 3MA inhibited the expression of Atg5, reduced LC3-II/LC3-I ratio, elevated the level of P62, decreased the colocalization of LD and LC3, reduced the outflow of cholesterol, increased the content of TC and CE, and elevated CE/TC ratio in foam cells （P<0.05）. CONCLUSION Lipophagy is enhanced at 24 h but decreased at 48 h during foam cell formation. Lipophagy inhibited foam cell formation by reducing cholesterol content and increasing cholesterol efflux.     

Ca2+-activated Cl- channels and pulmonary hypertension induced by high pulmonary blood flow

Pulmonary hypertension induced by high pulmonary blood flow involves a variety of complex mechanisms, including endothelial damage, pulmonary artery smooth muscle relaxation-contraction disorder and vascular remodeling. Besides, the factor of ion channels in pulmonary artery smooth muscle cells is also highly correlated to vasoconstriction. In recent years, many studies have shown that activation of Ca²⁺-activated Cl^- channels is responsible for the membrane depolarization of pulmonary artery smooth muscle cells, and plays an important role in the regulation of vascular tone and vasoconstriction. This article reviews the biophysical and pharmacological characteristics of Ca²⁺-activated Cl^- channels as well as the influence of Ca²⁺-activated Cl^- channels in high pulmonary blood flow-induced pulmonary hypertension.     

Qishen-Yiqi dripping pills attenuate atherosclerosis by regulating Ca2+ homeostasis via TRPC1/STIM1 pathway

AIM:To explore the therapeutic effect of Qishen-Yiqi dripping pills (QS) on atherosclerosis (AS) and the mechanism. METHODS:AS rat model was established by high-fat diet, and SD rats were randomly divided into normal control group, AS model group, low-dose, middle-dose and high-dose QS groups, and positive group (n=6 each). After administration for 12 weeks, serum samples were collected to detect the serum lipid and Ca²⁺ levels. HE staining was used evaluated the histopathological changes of arterial tissue. The serum levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were measured by ELISA. The nitric oxide (NO) level was detected by nitrate reductase method. The protein levels of transient receptor potential channel protein 1 (TRPC1), stromal interaction molecule 1 (STIM1) and endothelial NO synthase (eNOS) were determined by Western blot. RESULTS:QS significantly reduced the arterial damage via inhibiting the formation of atherosclerotic plaque and attenuated intimal thickening and vascular stenosis. Compared with AS group, the serum levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) were decreased significantly and the levels of high-density lipoprotein cholesterol (HDL-C) were increased significantly in high-dose QS group (P<0.05). The serum levels of IL-1β, IL-6 and TNF-α in high-dose QS group were lower than those in AS group (P<0.05). Compared with AS group, the serum Ca²⁺ level was lowered and the arterial tissue NO level was elevated in QS groups (P<0.05). Compared with AS rats, the protein levels of TRPC1 and STIM1 were decreased significantly and the protein level of eNOS was increased significantly in the rats treated with QS (P<0.05). CONCLUSION:QS regulate calcium homeostasis via TRPC1/STIM1 pathway, increase the production of NO and inhibit the inflammatory responses, thus exerting anti-AS effect.     

ROS mediates regulation of intracellular Ca2+ induced by angiotensin II in primarily cultured medullary neurons

AIM: To investigate the role of reactive oxygen species (ROS) in the regulation of intracellular Ca²⁺ induced by angiotensin II (Ang II) in the primarily cultured medullary neurons. METHODS: Primarily cultured medullary neurons were prepared from 14-day-old embryos of Sprague-Dawley rats in the study. The identification of medullary neurons was assessed by double-labeling immunofluorescence. To explore the role of ROS, mainly the superoxide (O₂^·), the O₂^·generation was measured using the fluorogenic probe dihydroethidium (DHE). To determine intracellular free calcium concentration ([Ca²⁺]_i), the neurons were loaded with the Ca²⁺-specific dye Fura-2/AM. The cell viability after adding Ang II was also examined using CCK-8 assay. RESULTS: Most of the cultured cells were medullary neurons, more than 80% of which were glutamate positive neurons. Ang II (5 μmol/L) increased the level of ROS within 10 min in the medullary neurons. Ang II at 5 μmol/L induced a significant[Ca²⁺]_i increase in the medullary neurons, and the effect of Ang II occurred rapidly and reached a peak within 20 min after administration. The level of[Ca²⁺]_i started to decline after washout. The Ca²⁺ elevation induced by Ang II was significantly decreased by apocynin or TEMPOL. No significant difference in the cell viability between control group and 5 μmol/L Ang II treatment group was observed. CONCLUSION: ROS is involved in the regulation of[Ca²⁺]_i induced by Ang II in the primarily cultured medullary neurons, suggesting a potential intracellular signaling mechanism involved in the Ang II-mediated oxidant regulation of central neural control of blood pressure.     

低温胁迫下北海道黄杨叶肉细胞Ca2+和Ca2+-ATPase的变化

 利用焦锑酸钙沉淀和硝酸铅沉淀的电镜细胞化学方法,以室温生长的北海道黄杨植株为对照,研究了人工4 ℃低温胁迫过程中北海道黄杨（Euonymus japonicus‘Cuzhi’）叶肉细胞Ca²⁺和Ca²⁺-ATPase的动态变化。在4 ℃低温胁迫的初期（3 ~ 12 h）,北海道黄杨叶肉细胞间隙和液泡内的Ca²⁺沉淀颗粒减少,而细胞质和细胞核内的Ca²⁺水平升高,但Ca²⁺-ATPase在细胞的分布几乎没有变化,主要分布在质膜和液泡膜上,有较高的活性;低温胁迫24 h,细胞质和细胞核内增加的Ca²⁺开始回到细胞间隙和液泡中,Ca²⁺-ATPase在质膜和液泡膜上活性增强;在低温胁迫48 ~ 96 h,细胞内的Ca²⁺又回到低温胁迫前的低水平,但Ca²⁺-ATPase在质膜和液泡膜上仍有很高的活性。叶肉细胞内Ca²⁺稳态平衡和Ca²⁺-ATPase的活性变化与植物的抗寒性存在一定的相关性。     

Effect of OX-LDL and simvastatin on PKC activity and cytosolic free Ca2+ in cultured rat aortic smooth muscle cells

AMI:To clarify whether OX-LDL and simvastatin can induce the changes of PKC activity and cytosolic free Ca²⁺ in rat aortic smooth muscle cells (ASMC). METHODS:PKC activity and cytosolic free Ca²⁺ were measured by its ability to transfer phosphate from [³²P]ATP to lysine-rich histone and flow cytometric analysis after loading with the Ca²⁺dye fluo-3/Am, respectively. RESULTS:OX-LDL increased PKC total activity in a dose-dependent manner and induced translocation of PKC from the cytosolic to membrane, while OX-LDL induced biphasic [Ca²⁺]i responses including the rapid initial transient phase and the sustained phase. When simvastatin was added, the translocation of PKC was markedly decreased and simvastatin did not impair the initial peak response to OX-LDL but significantly reduced the subsequent plateau phase. CONCLUSSION:OX-LDL can induce dynamic changes of signal transduction of PKC and cytosolic free Ca²⁺ in ASMC and these two events are closely linked.