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.     

Strontium ranelate promotes osteogenic differentiation of rat bone mesenchymal stem cells through TGF-β1/Smad signaling pathway

[ABSTRACT] AIM: To study the roles of transforming growth factor β1 (TGF-β1)/Smad signaling pathway in strontium ranelate (Sr)-induced osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). METHODS: In the process of osteogenic differentiation of rat BMSCs, the expression of phosphorylated Smad2 (p-Smad2) and Runx2 was detected by Western blotting after the cells were treated with Sr. BMSCs were pretreated with SB431542, a selective inhibitor of TGF-β1, or Smad2 small interfering RNA (Smad2-siRNA), followed by Sr treatment, and then the expression of p-Smad2 and Runx2 was observed. At the same time, the activity of alkaline phosphatase (ALP) and the level of calcium nodules were detected to determine the osteogenic differentiation of BMSCs. RESULTS: The expression levels of p-Smad2 and Runx2 were enhanced under the action of Sr in the process of osteogenic differentiation of rat BMSCs. The expression of p-Smad2 reached to maximum when BMSCs were treated with Sr at concentration of 1 mmol/L for 1 h. The expression of Runx2 reached to maximum when BMSCs were treated with Sr at concentration of 1 mmol/L for 5 d. The pretreatment with SB431542 or Smad2-siRNA inhibited not only the expression of p-Smad2 and Runx2, but also the activity of ALP and the level of calcium nodules. CONCLUSION: Sr promotes the osteogenic differentiation of rat BMSCs through the TGF-β1/Smad signaling pathway.     

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.     

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.     

Mechanisms of catalpol-induced osteogenic differentiation in SD rat bone marrow mesenchymal stem cells

AIM: To investigate the mechanisms for catalpol-induced osteogenic differentiation of SD rat bone marrow mesenchymal stem cells (BMSCs) in vitro. METHODS: The cells were divided into control group, osteoinduction group and catalpol group. The activity of alkaline phosphatase (ALP) was measured at 7 d, 14 d and 21 d after catapol treatment, meanwhile ALP positive cell numbers and calcium nodes were counted at 14 d and 21 d after catapol treatment,respectively. The mRNA expression of Runx2, osteocalcin, Wnt3a, β-catenin, Wnt5a and Wnt11 was detected at 7 d, 14 d and 21 d after catapol treatment by real-time PCR. RESULTS: Catalpol at 2.0 mg/L increased ALP activity and ALP positive cell numbers significantly(P<0.05), meanwhile, it also increased calcium nodes numbers in cultured BMSCs (P<0.05). Compared with control group, catalpol increased the mRNA expression of Runx2 significantly at 14 d, but not at the 7 d and 21 d. Catapol also promoted the mRNA expression of osteocalcin significantly from 7 d to 21 d. Compared with control group, the mRNA expression of Wnt3a and β-catenin in catalpol group was increased at 14 d and 21 d. In addition, the mRNA expression of Wnt5a and Wnt11 in catalpol group was higher than that in control group at 14 d, but that was decreased at 21 d. CONCLUSION: Catalpol induces differentiation of BMSCs into osteoblast by increasing the mRNA expression of Runx2, and promotes the differentiation and mature of these osteoblasts by increasing ALP secretion, osteocalcin mRNA expression and calcium deposition. The activation of Wnt signaling pathway may be involved in this pro-osteogenic differentiation process.     

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.     

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.     

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.     

Strontium renelate promotes osteogenesis via regulation of Sonic hedgehog signaling molecules in rat bone marrow mesenchymal stem cells

AIM：To study the role of Sonic Hedgehog (Shh) in strontium ranelate (Sr)-induced osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). METHODS：BMSCs was isolated from 4-week-old rats by adherent culture. The cells in the 3rd～5th generations were induced to differentiate into obteoblasts, and then were treated with different concentrations of Sr and cyclopamine (Cy). The activity of alkaline phosphatase (ALP) was mea-sured by colorimetry. Mineralized nodules were observed by alizarin red staining. The cellular Shh and Runx2 expression was detected by Western blotting. RESULTS：Sr at concentration of 3 mmol/L increased the activity of ALP and induced the formation of mineralized nodules. Sr at concentrations ranging from 0.1 to 5 mmol/L increased the expression of Shh and Runx2 in the BMSCs at 7 d. Furthermore, the peak expression of Shh occurred following the exposure of Sr (1 mmol/L) or Runx2 (3 mmol/L). On the other hand, Sr at concentration of 1 mmol/L showed a time-dependent increase in the expression of Shh and Runx2 from 1 d to 7 d. Cy at concentration of 10 μmmol/L not only obviously inhibited Sr-induced expression of Shh and Runx2, but also antagonized the increase in the ALP activity and mineralization induced by Sr in the BMSCs. CONCLUSION：Sr promotes osteogenic differentiation of BMSCs by increasing the expression of Shh and Runx2.     

Alteration of myocardial sarcoplasmic reticulum Ca2+ transport protein expression in neonatal hypothyroid rats

AIM: To investigate the alteration of sarcoplasmic reticulum (SR) Ca²⁺ transport proteins including sarcoplasmic reticulum Ca²⁺-ATPase 2a(SERCA2a) and phospholamban(PLB) mRNA expression as well as the alteration of myocardial SR Ca²⁺-ATPase activity in neonatal hypothyroid rats, and to explore the effect of levothyroxine(L-T₄) substitution therapy on the above indexes.METHODS: Hypothyroidism was induced by the administration of propylthiouracil (PTU, 50 mg/d) to the pregnant SD rats by gavage beginning on embryonic day 15 and continuing throughout the lactational period. A subgroup of neonatal hypothyroid rats were intraperitoneally injected with L-T₄ levothroxine (20 μg/kg BW daily), starting from the day of birth. Other pregnant SD rats received normal saline instead of PTU. The samples of the rats in all 3 groups were harvested at postnatal day 3, 5 and 7 respectively (n=10). After measurement of serum thyroid hormone levels, the hearts were removed and the ventricles were weighed (HW). The concentration of calcium in ventricular myocardium(ventricular myoCa²⁺) was detected by fluorospectrophotometry and the activity of SR Ca²⁺-ATPase was determined by the inorganic phosphorus method. The mRNA expression of SERCA2a and PLB was also detected by real-time PCR. RESULTS: Neonatal hypothyroid rats had a significant lower level of SERCA2a mRNA (P<0.05) and a higher level of PLB mRNA (P<0.01), and subsequent lower SERCA2a/PLB at each postnatal day (P<0.01) was observed. Compared with hypothyroid group, the mRNA expression of SERCA2a significantly increased (P<0.05) and that of PLB significantly decreased (P<0.05) in L-T₄ treatment group. The concentration of ventricular MyoCa²⁺ in hypothyroid group was significantly higher than that in control group (P<0.01), and that in L-T₄ treatment group showed a significant decrease as compared with hypothyroid group (P<0.05). The activity of sarcoplasmic reticulum Ca²⁺-ATPase in hypothyroid group was significantly lower than that in control group (P<0.01), and that in L-T₄ treatment group showed a significant increase as compared to hypothyroid group (P<0.05). CONCLUSION: The deficiency of thyroid hormone, resulting in decreased expression of SERCA2a mRNA as well as increased PLB mRNA, contributes to the reduction of SR Ca²⁺-ATPase activity in neonatal rats. This may be one of the most important mechanisms of myocardial systolic and diastolic dysfunctions.     

Effects of Ca2+on the releases of ET-1, NO and PGI2 from bovine coronary artery endothelial cells during hypoxia

AIM: To explore the mechanism of ET-1, NO and PGI² release from coronary artery endothelial cells(CAEC) induced by acute hypoxia. METHODS: Bovine coronary artery endothelial cells were cultured and [⁴⁵ Ca²⁺] was used to investigate the difference of calcium uptake between normoxia group and hypoxia group(3% O₂). The contents of ET-1, NO and PGI² in media of normoxia group, hypoxia group and hypoxia + verapamil group were measured 24 h after hypoxia. RESULTS: [ ⁴⁵ Ca²⁺] uptake by CAEC in hypoxia group was 1.9 times more than normoxia group(P< 0.01). Hypoxia + verapamil group released more PGI², ET-1 and less NO than hypoxia group(P< 0.05). CONCLUSION: Changes of ET-1, NO and PGI² releases during hypoxia may be caused by the inflow of Ca²⁺ into coronary artery endothelial cells.     

The roles of ERK signaling pathway in osteogenic differentiation of rat MSCs promoted by quercetin

AIM:To study the roles of extracellular signal-regulated kinase(ERK) signal pathway in the process of osteogenic differentiation in rat mesenchymal stem cells(MSCs) promoted by quercetin(QUE). METHODS:The optimal concentration of QUE for promoting osteogenic differentiation of rat MSCs was determined by MTT and alkaline phosphatase(ALP) detection. The activity of ALP was detected by the ALP detection kit. The expression of bone Gla protein(BGP) and collagen typeⅠ(ColⅠ) was observed by ELISA analysis. MSCs were exposed to QUE at optimal concentration with or without ERK1/2 inhibitor PD98059. Non-phosphorylated and phosphorylated expression of ERK1/2 was analyzed by Western blotting. The mRNA expression of transforming growth factor β₁(TGF-β₁), bone morphogenetic protein 2(BMP-2) and core binding factor α1(Cbfα1) was measured by fluorescence quantitative PCR. RESULTS:QUE at concentrations of 0.1 μmol/L, 1 μmol/L and 10 μmol/L induced the expression of ALP in MSCs in a dose-dependent manner, and also promoted MSCs proliferation. The expression levels of ALP, BGP and ColⅠwere higher in QUE group, and was lower in PD89059 group than those in control group. Compared with control group, the level of phosphorylated ERK1/2, and the mRNA expression of TGF-β₁, BMP-2 and Cbfα1 increased in QUE group. The mRNA expression of TGF-β₁, BMP-2 and Cbfα1 in QUE+PD98059 group decreased as compared with QUE group. CONCLUSION:QUE promotes osteogenic differentiation of MSCs by activating ERK signaling pathway.     

Inhibitory effect of ATP on proliferative signaling in immortalized human fibroblasts

AIM: To investigate the inhibitory effects of ATP on proliferation signaling in immortalized human fibroblasts. METHODS: Immortalized human fibroblasts were treated with ATP, ATP conbined with calcium or potassium channel antagonists, respectively. The intracelluar-free calcium ([Ca²⁺]i), inositol 1,4,5-trisphosphate(IP₃) levels and cell viability were detected at different time points. RESULTS: ATP significantly increased the [Ca²⁺]i and decreased the IP₃ level in immortalized human fibroblasts, especially at initial stage (P＜0.01) . Compared to ATP alone, the proliferation rates remarkably increased when calcium or potassium channel antagonists were used (P＜0.01, respectively) with ATP. CONCLUSION: The calcium and potassium channels and IP₃ involved in the inhibitory effects of ATP on the proliferative signaling in immortalized human fibroblasts.     

Effects of lentivirus-mediated transfection of shRNA targeting Adra1d gene on calcium and calmodulin in rat aortic vascular smooth muscle cells

AIM:To investigate the effects of lentivirus-mediated transfection of shRNA targeting α_1D-adrenergic receptor (Adra1d) gene on calcium ion (Ca²⁺) and calmodulin (CaM) in vascular smooth muscle cells (VSMCs) of rat aorta. METHODS:Single oligonucleotide sequences of shRNA targeting rat Adra1d gene were design and synthesized, and then the shRNA was constructed and cloned into GV248 vector. The U6-shRNA carrier and expression vector were transfected into 293T cells together and packed with lentivirus, and the supernatant was collected and concentrated by overspeed centrifugation. The VSMCs of rat aorta were transfected with recombinant lentivirus vector. The interference effects were identified by RT-qPCR and Western blot. The concentration of Ca²⁺ in VSMCs was detected by laser confocal inspection, and the expression of CaM at mRNA and protein levels in the VSMCs was determined by RT-qPCR and Western blot. RESULTS:The lentiviral shRNA expression vector was successfully constructed. The titer of the concentrated virus was 3×10¹¹ TU/L. The mRNA and protein expression levels of Adra1d in the rat aortic VSMCs were significantly reduced after transfection. The interference efficiency of Lv-shRNA4-Adr to Adrald gene was greater than 85%. After target silencing of Adra1d gene, compared with scrambled group, the Ca²⁺ fluorescence intensity of rat aortic VSMCs was significantly increased. Moreover, the mRNA and protein expression levels of CaM were also increased significantly. CONCLUSION:A lentiviral shRNA expression vector targeting rat Adra1d gene was successfully constructed, which significantly increased Ca²⁺ concentration and CaM expression in rat aortic VSMCs.     

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.     

Calcium overload and reactive oxygen species mediate high glucose-induced apoptosis of mouse osteoblast MC3T3-E1 cells

AIM: To investigate the role of reactive oxygen species (ROS) and calcium overload in the apoptosis of MC3T3-E1 cells induced by high glucose. METHODS: Cultured mouse skull bone-derived osteoblast cell line MC3T3-E1 was treated with high concentration of D-glucose to induce apoptosis. The proliferation of MC3T3-E1 cells was detected by MTT assay after treated with different concentrations of D-glucose for 24 h and 48 h. The apoptotic rate and the intracellular levels of calcium and ROS were also measured after the cells were treated with high glucose (35 mmol/L) for 24 h. RESULTS: After high glucose treatment, the cell proliferation was inhibited. The early apoptosis and total cell death increased to (24.16?3.53)% and (63.74?4.32)%,respectively. High glucose treatment significantly increased intracellular levels of ROS and Ca²⁺. The increased apoptotic rate was reduced by addition of antioxidant N-acetylcysteine and calcium chelator BAPTA-AM. Inhibition of store-operated Ca²⁺ channels by La³⁺ also decreased the intracellular level of Ca²⁺ and cell apoptosis induced by high glucose. CONCLUSION: High glucose increases intracellular ROS level and the release of Ca²⁺ through the store-operated Ca²⁺ channels, thus resulting in intracellular Ca²⁺ overload and leading to apoptosis of osteoblasts.     

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.     

Propofol inhibits LPS-induced inflammatory response in microglia via TRPV1 ion channels

AIM:To investigate the effects of propofol (P) on the inflammatory response of microglia induced by lipopolysaccharide (LPS) and the mechanisms. METHODS:Mouse microglia BV2 cells were treated with LPS at 100 μg/L to establish a neuroinflammatory injury model. The BV2 cells were divided into 4 groups:control group (C group), model group (L group), L+P group and LPS+AMG517 group (L+A group). The level of tumor necrosis factor-α (TNF-α) in the cell culture supernatant was measured by ELISA. The mRNA expression of transient receptor potential cation channel subfamily V member 1 (TRPV1) was detected by real-time PCR. The protein levels of TRPV1, TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and phosphorylated calcium/calmodulin-dependent protein kinase Ⅱ (p-CaMKⅡ) were determined by Western blot. The content of free Ca²⁺ in the microglia BV2 cells was detected by Fluo-3 AM assay. RESULTS:Compared with C group, the level of TNF-α was significantly increased in L group (P<0.01), but that in P group was not changed. Compared with L group, the level of TNF-α was significantly lower than that in L+P group within 4 h (P<0.01). Compared with C group, the mRNA expression of TRPV1 was significantly increased in L group (P<0.01). Compared with L group, the mRNA expression of TRPV1 was significantly down-regulated in L+P group (P<0.01).Compared with L group, the protein levels of TNF-α, IL-1β, IL-6 and p-CaMKⅡ and intracellular Ca²⁺ concentration were significantly lower than those in L+P group and L+A group (P<0.01). CONCLUSION:Propofol inhibits the inflammatory response of microglia by reducing the expression of TNF-α, IL-1 and IL-6, which may be related to the down-regulation of TRPV1 and p-CaMKⅡ and the reduction of intracellular Ca²⁺ concentration.