Effect of electroacupuncture on calcium homeostasis in hippocampal neurons of mice with sepsis-associated encephalopathy / 中华麻醉学杂志

Objective:To investigate the effect of electroacupuncture on calcium homeostasis in hippocampal neurons of mice with sepsis-associated encephalopathy (SAE).Methods:Twenty-four healthy male C57BL/6J mice, weighing 18-22 g, were divided into 4 groups ( n=6 each) using a random number table method: sham operation group (Sham group), SAE group, SAE plus electroacupuncture group (SAE+ EA group), and SAE plus sham electroacupuncture group (SAE+ SEA group). The virus carrying calcium ion (Ca 2+ ) fluorescent probes was injected and then an optical fiber was implanted into the hippocampal CA1 area to record the fluorescence signals of Ca 2+ . SAE was induced by cecal ligation and puncture in anesthetized mice at 3 weeks after administration. Starting from 3 days before surgery, Baihui and bilateral Quchi and bilateral Zusanli acupoints were stimulated for 30 min per day for 7 consecutive days in SAE+ EA group. In SAE+ SEA group, electroacupuncture was performed at the points 0.2 mm lateral to the corresponding acupoints without electrical stimulation. Open field tests were conducted at 5 days after surgery to record the number of rearing and changes in related Ca 2+ signals in hippocampal CA1 neurons. Novel object recognition tests were conducted at 6-7 days after surgery to record the recognition time and changes in related Ca 2+ signals in hippocampal CA1 neurons. Mice were sacrificed after the end of behavioral testing on 7 days after surgery, and brain tissues ipsilateral to the optical fiber implant were obtained and the fluorescence intensity of Ca 2+ in the hippocampal CA1 neurons was acquired using a fluorescent microscope. Results:Compared with Sham group, the number of rearing and amplitudes of related Ca 2+ signals in hippocampal CA1 neurons while rearing were significantly decreased in SAE group and SAE+ SEA group ( P<0.05), and no statistically significant changes were found in the parameters mentioned above in SAE+ EA group ( P>0.05), and the recognition index and amplitudes of related Ca 2+ signals while recognizing were significantly deceased, and the fluorescence intensity of Ca 2+ in hippocampal CA1 neurons was increased in SAE, SAE+ EA and SAE+ SEA groups ( P<0.05). Compared with SAE group and SAE+ SEA group, the number of rearing and amplitudes of related Ca 2+ signals in hippocampal CA1 neurons while rearing were significantly increased, the recognition index and amplitudes of related Ca 2+ signals in hippocampal CA1 neurons while recognizing were increased, and the fluorescence intensity of Ca 2+ in hippocampal CA1 neurons was decreased in SAE+ EA group ( P<0.05). There were no statistically significant differences in the parameters mentioned above between SAE group and SAE+ SEA group ( P>0.05). Conclusions:The mechanism by which electroacupuncture alleviates SAE may be related to regulation of Ca 2+ homeostasis in hippocampal neurons of mice.

KCTD4 interacts with CLIC1 to disrupt calcium homeostasis and promote metastasis in esophageal cancer

Increasing evidences suggest the important role of calcium homeostasis in hallmarks of cancer, but its function and regulatory network in metastasis remain unclear. A comprehensive investigation of key regulators in cancer metastasis is urgently needed. Transcriptome sequencing (RNA-seq) of primary esophageal squamous cell carcinoma (ESCC) and matched metastatic tissues and a series of gain/loss-of-function experiments identified potassium channel tetramerization domain containing 4 (KCTD4) as a driver of cancer metastasis. KCTD4 expression was found upregulated in metastatic ESCC. High KCTD4 expression is associated with poor prognosis in patients with ESCC and contributes to cancer metastasis in vitro and in vivo. Mechanistically, KCTD4 binds to CLIC1 and disrupts its dimerization, thus increasing intracellular Ca2+ level to enhance NFATc1-dependent fibronectin transcription. KCTD4-induced fibronectin secretion activates fibroblasts in a paracrine manner, which in turn promotes cancer cell invasion via MMP24 signaling as positive feedback. Furthermore, a lead compound K279-0738 significantly suppresses cancer metastasis by targeting the KCTD4‒CLIC1 interaction, providing a potential therapeutic strategy. Taken together, our study not only uncovers KCTD4 as a regulator of calcium homeostasis, but also reveals KCTD4/CLIC1-Ca2+-NFATc1-fibronectin signaling as a novel mechanism of cancer metastasis. These findings validate KCTD4 as a potential prognostic biomarker and therapeutic target for ESCC.

Research progress on hypoxia-induced imbalance of calcium homeostasis in pulmonary artery smooth muscle cells and its treatment / 中国药理学通报

Chronic hypoxic lung diseases are major causes of disability and mortality worldwide, which are typically aggravated by hypoxic pulmonary hypertension.The pathogenesis of hypoxic pulmonary hypertension is complex, and its mechanism has not been fully elucidated.The previous studies have shown abnormally elevated levels of free Ca + in the cytoplasm of pulmonary artery smooth muscle cells to be the predominant drivers of pulmonary hypertension , causing continuous contraction and remodeling of the pulmonary vessels.This article briefly summarizes the mechanism of hypoxia-induced imbalance in calcium homeostasis in pulmonary artery smooth muscle cells, together with its related drug research, based on the existing literature.Hypoxia induces an imbalance in calcium homeostasis in pulmonary artery smooth muscle cells by regulating hypoxia-inducible factor-1, K+ , store-operated calcium channel, receptor-operated calcium channel, the Ca +-sensing myosin contractile mechanism by binding to calmodulin, leading to pulmonary vasoconstriction.Ca + can also activate PKC/ MAPKs and PI3K/Akt/mTOR pathways, leading to pulmonary vascular remodeling.

IRE1α deficiency impairs autophagy in chondrocytes by upregulating calcium homeostasis endoplasmic reticulum protein / 南方医科大学学报

OBJECTIVE@#To explore the mechanism by which inositol-requiring enzyme-1α (IRE1α) regulates autophagy function of chondrocytes through calcium homeostasis endoplasmic reticulum protein (CHERP).@*METHODS@#Cultured human chondrocytes (C28/I2 cells) were treated with tunicamycin, 4μ8c, rapamycin, or both 4μ8c and rapamycin, and the expressions of endoplasmic reticulum (ER) stress- and autophagy-related proteins were detected with Western blotting. Primary chondrocytes from ERN1 knockout (ERN1 CKO) mice and wild-type mice were examined for ATG5 and ATG7 mRNA expressions, IRE1α and p-IRE1α protein expressions, and intracellular calcium ion content using qPCR, Western blotting and flow cytometry. The effect of bafilomycin A1 treatment on LC3 Ⅱ/LC3 Ⅰ ratio in the isolated chondrocytes was assessed with Western blotting. Changes in autophagic flux of the chondrocytes in response to rapamycin treatment were detected using autophagy dual fluorescent virus. The changes in autophagy level in C28/I2 cells overexpressing CHERP and IRE1α were detected using immunofluorescence assay.@*RESULTS@#Tunicamycin treatment significantly up-regulated ER stress-related proteins and LC3 Ⅱ/LC3 Ⅰ ratio and down-regulated the expression of p62 in C28/I2 cells (P < 0.05). Rapamycin obviously up-regulated LC3 Ⅱ/LC3 Ⅰ ratio (P < 0.001) in C28/I2 cells, but this effect was significantly attenuated by co-treatment with 4μ8c (P < 0.05). Compared with the cells from the wild-type mice, the primary chondrocytes from ERN1 knockout mice showed significantly down-regulated mRNA levels of ERN1 (P < 0.01), ATG5 (P < 0.001) and ATG7 (P < 0.001), lowered or even lost expressions of IRE1α and p-IRE1α proteins (PP < 0.01), and increased expression of CHERP (P < 0.05) and intracellular calcium ion content (P < 0.001). Bafilomycin A1 treatment obviously increased LC3 Ⅱ/ LC3 Ⅰ ratio in the chondrocytes from both wild-type and ERN1 knockout mice (P < 0.01 or 0.05), but the increment was more obvious in the wild-type chondrocytes (P < 0.05). Treatment with autophagy dual-fluorescence virus resulted in a significantly greater fluorescence intensity of LC3-GFP in rapamycin-treated ERN1 CKO chondrocytes than in wild-type chondrocytes (P < 0.05). In C28/I2 cells, overexpression of CHERP obviously decreased the fluorescence intensity of LC3, and overexpression of IRE1α enhanced the fluorescence intensity and partially rescued the fluorescence reduction of LC3 caused by CHERP.@*CONCLUSION@#IRE1α deficiency impairs autophagy in chondrocytes by upregulating CHERP and increasing intracellular calcium ion content.

Mitochondrial Oxidative Stress Enhances Vasoconstriction by Altering Calcium Homeostasis in Cerebrovascular Smooth Muscle Cells under Simulated Microgravity / 生物医学与环境科学（英文）

Objective@#Exposure to microgravity results in postflight cardiovascular deconditioning in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been reported during this process. To elucidate the mechanism for this condition, we investigated whether mitochondrial oxidative stress regulates calcium homeostasis and vasoconstriction in hindlimb unweighted (HU) rat cerebral arteries.@*Methods@#Three-week HU was used to simulate microgravity in rats. The contractile responses to vasoconstrictors, mitochondrial fission/fusion, Ca @*Results@#An increase of cytoplasmic Ca @*Conclusion@#The present results suggest that mitochondrial oxidative stress enhances cerebral vasoconstriction by regulating calcium homeostasis during simulated microgravity.

Protective effect of iridoid glycosides of radix scrophulariae on endoplasmic reticulum stress induced by oxygen-glucose deprivation and reperfusion / 浙江大学学报·医学版

OBJECTIVE@#To investigate the regulatory effect of iridoid glycoside of radix scrophulariae (IGRS) on endoplasmic reticulum stress induced by oxygen-glucose deprivation and reperfusion @*METHODS@#Rat pheochromocytoma PC12 cells were pretreated with IGRS (50, 100, 200 μg/mL) for 24h, and the @*RESULTS@#The damage caused by OGD/R to PC12 cells was significantly reduced by IGRS, with significant effect on increasing survival rate and reducing LDH release (all @*CONCLUSIONS@#IGRS has neuroprotective effect, which may alleviate cerebral ischemia-reperfusion injury by regulating SERCA2, maintaining calcium balance, and inhibiting endoplasmic reticulum stress-mediated apoptosis.

Role of Calcium Toxicity in Neurodegenerative Diseases

Background: Neurodegenerative diseases are those affect the central nervous system (brain, spinal cord) and peripheral nervous system. It is characterized by progressive loss of neurons and synapses in these nervous systems. Calcium homeostasis receives key attention in the past few years in the field of neuronal physiology of Ageing and Neurodegenerative diseases. Objective: The aim of this study was to evaluate Calcium homeostasis in patients with Neurodegenerative diseases. Methods: 50 subjects (36 males and 14females) with Neurodegenerative diseases. And same number of healthy age-matched subject control group was assessed. A complete & detailed neurological examination were performed in all individuals and clinically evaluated for the occurrence of Neurodegenerative diseases. Calcium levels were estimated using (Instrumentation Laboratory – IlyteTM) Automated Electrolyte Analyzer, with solution pack and reagents. Results: ‘The Serum Calcium (Ca2+) levels were estimated and comparison was done in both control and case group separately, for evaluating the changes due to ageing and in particular to neurodegenerative diseases. Conclusion: Neurodegenerative diseases showed significant differences in Calcium homeostasis.

Genes related to catecholaminergic polymorphic ventricular tachycardia / 国际儿科学杂志

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a kind of common hereditary arrhythmia syndrome in adolescents.Its core is arrhythmia induced by adrenaline,and the root underlying cause of the arrhythmia is rapid ventricular tachycardia (bidirectional or polymorphic).The CPVT patient often has no obvious clinical manifestations,and almost normal in examinations,which may lead to missed diagnosis and misdiagnosis.Gene detection may be a classical and reliable diagnostic method.It has been found that mutation genes related to CPVT include Ryanodine receptor 2,Calsequestrin 2,and their mutations affect calcium homeostasis,causing electrophysiological abnormalities of cardiomyocytes,overloading calcium ions in cardiomyocytes,leading to the delayed depolarization of cardiomyocytes and further induction of ventricular arrhythmias.In this review,we summarize the mutations of ryanodine receptor 2,calsequestrin 2 and other possible genes related to CPVT,which is helpful for clinical precise treatment and exploration of the molecular mechanism of CPVT.

Effects of renal Na-Cl co-transporter on calcium homeostasis and bone metabolism / 中华临床营养杂志

Osteoporosis is a global public health problem. The kidney, especially the Na-Cl co-trans-porter ( NCC) located in the renal distal tubular cells, which is an important transporter for urinary calcium reg-ulation, plays an important role in calcium homeostasis and bone metabolism. This review summarized recent re-searches on effects and mechanisms of NCC on calcium and bone metabolism.

A homozygous mutation in TMEM38B causes rare osteogenesis imperfecta type XIV / 基础医学与临床

Objective To investigate the phenotype of a boy with osteogenesis imperfecta(OI)and detect the path-ogenic gene mutation in his family.Methods The clinical data of a uygur ethnic boy was investigated in detail, who suffered from early onset repeated fragile fractures.Bone turnover biomarkers, bone mineral density(BMD) and bone morphology were evaluated.The pathogenic mutations in this patient were investigated by targeted next-generation sequencing and subsequently confirmed by Sanger sequencing.Results Serum β-cross linked C-te-lopeptide of type Ⅰcollagen was elevated.Radiological assessment revealed a generalized osteoporosis in thoraco-lumbar spine,slender long bone with thin cortices.The pathogenic mutations in TMEM38B were detected as follow:a homozygous mutation c.507G>A transition in exon 4,which would generate a new downstream termination codon (p.W169X).His parents were heterozygous carriers of the mutation.Conclusions Mutation in TMEM38B is iden-tified for the first time in a uygur ethnic boy with extremely rare autosomal recessive OI type XIV.The clinical and genetic findings expands our understanding of rare OI induced by TMEM38B mutation.

Calcium homeostasis modulator 1 ( CALHM1 ) polymorphisms in cattle

The calcium homeostasis modulator 1 gene (CALHM1), which is located on chromosome 10 in humans and on chromosome 26 in cattle, is a transmembrane glycoprotein that controls the cytosolic calcium concentrations. Altered calcium homeostasis has been associated with several neurodegenerative disorders, including Alzheimer's disease (AD). In a recent study, single nucleotide polymorphisms (SNPs) of CALHM1 have been associated with sporadic Creutzfeldt-Jakob disease (CJD). The protein sequence of human CALHM1 shows 93% homology with bovine CALHM1. Although SNPs of human CALHM1 have been correlated with human prion disease, polymorphisms of the bovine CALHM1 gene have not been reported in cattle thus far. To investigate polymorphisms of the bovine CALHM1 gene in Korean native cattle, we analyzed the open reading frame (ORF) of this gene in 175 Hanwoo and 141 Holstein cattle. We observed five SNPs: c.219C>T (rs380966453), c.357C>T (rs385969338), and c.869A>G (rs516301908) within the ORF region of two exons; and c.552+92A>G (rs481706737) and c.553-3A>C (rs448524869) in the intron of bovine CALHM1. Among the three SNPs that are in the ORF region of bovine CALHM1, the genotype and allele frequencies of the c.869A>G (p.His290Arg) and c.219C>T (p.Asn73Asn) SNPs were significantly different between Hanwoo and Holstein cattle (P<0.0001). Haplotype analysis showed that haplotypes ht2, ht3 and ht5 were also significantly different in these two cattle breeds. This study provides the first genetic analysis of the bovine CALHM1 gene in cattle.(AU)

Advances in the study of SORCIN in cancer drug resistance / 药学学报

Soluble resistance-related calcium-binding protein, SORCIN, is a 22 kDa calcium binding protein with "penta-EF hand", which participates in the regulation of intracellular calcium homeostasis in cells. SORCIN is highly expressed in many tissues such as hearts and brains. It is overexpressed in some of cancer tissues as well. Recently, a large amount of clinical data showed that SORCIN was closely related to drug resistance in cancer. Meanwhile, basic research found that SORCIN participates in the formation of multidrug resistance (MDR) and is related to severity and poor prognosis of tumors. Moreover, it may also regulate MDR induced by ATP-binding cassette transporters. Therefore, SORCIN is expected to become a new target for diagnosis and treatment of MDR. The present review summarizes recent progress in SORCIN study and its effect on MDR.

Progress of calcium signaling in sensory neurones and neuroglial cell in diabetic peripheral neuropathic dysfunction / 中国药理学通报

Ca2+signaling is fundamental for information process-ing in the peripheral nervous system,which regulates a variety of physiological activities.Ca2+signaling and calcium homeostasis are directly associated with neuropathology.Recently,studies on Ca2+signaling contribute to a deeper comprehension of the path-ogenesis of diabetic peripheral neuropathies,which provide a new research direction for the treatment of diabetic peripheralneuropathies.This review aims to highlight the relationship be-tween calcium signaling,sensory neurones and neuroglial cells in the context of diabetic peripheral neuropathies.

Zinc oxide nanoparticles inhibits Ca2+-ATPase1 expression induced by UVB irradiation in human lens epithelial cells / 眼科新进展

Objective To investigate the effect of ZnO nanoparticles on the expressions of plasma membrane calcium ATPasel (PMCA1) of human lens epithelial cell B-3 (HLEB-3) at both mRNA and protein levels in the presence and absence of ultraviolet B (UVB) irradiation.Methods HLEB-3 was cultured in RPMI 1640 medium,and the cytotoxic effect of different concentrations of ZnO (0 μg · mL-1,2.5 μg · mL-1,5.0μg · mL-1,10.0 μg · mL-1) on HLEB-3 was investigated in the presence and absence of UVB irradiation.DAPI staining was used to monitor the effect of ZnO on HLECB-3 nuclei,and cell apoptosis was evaluated using annexin V-FITC/PI staining in the presence and absence of UVB irradiation.In addition,the intracellular calcium ion (Ca2 +)levels were assayed using Fluo-3/AM staining,and the expression levels of both PMCA1 mRNA and protein within HLEB-3 were detected by real-time PCR and Western blot,respectively.Results DAPI staining showed that the ZnO-treated HLEB-3 displayed a concentration-dependent apoptosis,and UVB irradiation could further aggravate the cytotoxic effect of ZnO on HLEB-3.In addition,in the presence of UVB irradiation,concentration gradient of ZnO (2.5 μg · mL-1,5.0 μg · mL-1,10.0 μg · mL-1) increased the intracellular calcium ion levels [from (156.34 ±4.59) nmol · L-1 to (173.88 ±7.17)umol · L-1,(289.02 ± 9.09) nmol · L-1,(488.36 ± 48.16) nmol · L 1,respectively] and upregulated HLEB-3 apoptosis,with statistical difference (all P ＜ 0.05).Moreover,the expression level of PMCA1 in the 2.5 μg · mL-1,5.0 μg · mL-1,10.0 μg · mL-1 ZnO-treated epithelial cells was accordingly 0.75,0.57 and 0.41 as much as that in the 0μg · mL-1 ZnO-treated cells in the absence of UVB irradiation (all P ＜ 0.05),and was accordingly 0.64,0.24 and 0.09 in the present of UVB irradiation,with significant difference (all P ＜ 0.05).Conclusion Both ZnO nanoparticle and UVB irradiation can exert cosuppression effect on HLEB-3 via calcium-mediated signaling pathway,indicating it has great potential for the treatment of posterior capsular opacification with UVB irradiation.

Citotoxicity of Fipronil on Hepatocytes Isolated from Rat and Effects of Its Biotransformation

ABSTRACT The aim of this study was to characterize the mechanism of toxicity of fipronil on hepatocytes isolated from the rat and the effect of its biotransformation on the toxicological potential. The toxicity of fipronil was assessed by monitoring the oxygen consumption and mitochondrial membrane potential, intracellular ATP concentration, Ca2+ homeostasis and cell viability. The cell viability was evaluated by trypan blue exclusion in hepatocytes that were isolated from the normal rats and by the release of the enzymes alanine transaminase and aspartate transaminase in hepatocytes that were isolated from the normal rats or proadifen-pretreated rats. Fipronil reduced mitochondrial respiration in the cells that were energized with glutamate plus malate in a dose-dependent manner and dissipated the mitochondrial membrane potential that was accompanied by a reduction in ATP concentration and a disruption of intracellular Ca2+ homeostasis. The cell viability was affected by fipronil with higher potency in hepatocytes that were isolated from the normal rats, which indicated that the metabolism of this insecticide increased its toxicological potential. The results of this study indicated that the toxicity of fipronil to the hepatocytes was related to the inhibition of mitochondrial activity, which led to decreased ATP synthesis and a consequent alteration in intracellular Ca2+ homeostasis and ultimately resulted in cell death.

Research progress of Fructus Ligustri Lucidi and its compounds：effects on Bone and Mineral Metabolism / 中国药理学通报

Fructus Ligustri Lucidi ( FLL) is a traditional Chinese medicine commonly used in clinic. This paper reviews the phar-macological action of FLL and its compounds, especially demon-strates the regulation and mechanism in calcium homeostasis and bone metabolism.

Effects of lentivirus-mediated heat shock protein 70 gene on calcium homeostasis in PC12 cells undergone ischemia and hypoxia / 中华危重病急救医学

ObjectiveTo investigate the effects of lentivirus-mediated heat shock protein 70 (HSP70) gene on calcium homeostasis in PC12 cells undergone ischemia and hypoxia, and the mechanism involved.Methods PC12 cells at logarithmic phase were collected, and were divided into recombination lentivirus infection group (infected by lentivirus containing HSP70 and fluorescent gene), lentivirus control group (infected by lentivirus containing fluorescin without HSP70 gene) and non-infection group. HSP70 gene and protein expressions in PC12 cells were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot. After being challenged with ischemia and hypoxia for 4 hours, the viability of cells was detected by methyl thiazolyl tetrazolium (MTT), the levels of lactic acid dehydrogenase (LDH) in cell supernatant were determined by LDH measurement test kit. The concentration of intracelluer calcium ([Ca2+]i) was assayed by flow cytometer. The activities of Na+-K+-ATPase, Ca2+-Mg2+-ATPase and total ATPase were measured by ATPase test kits.Results The expressions of exogenous HSP70 gene and protein were found by RT-PCR and Western Blot in the recombination lentivirus infection group. After being challenged with ischemia and hypoxia, the viability of cells in the recombination lentivirus infection group was increased significantly as compared with the lentivirus control group and non-infection group (A value: 0.575±0.020 vs. 0.395±0.014, 0.363±0.045,t1= 17.996,t2= 10.600, bothP< 0.001), the levels of LDH in culture medium and the concentration of [Ca2+]i were decreased significantly [LDH (U/L): 743.46±23.68 vs. 935.43±34.77, 962.89±26.68,t1= 11.179, t2= 15.044, bothP< 0.001; [Ca2+]i relative fluorescence: (31.60±2.43)% vs. (41.48±3.33)%, (40.40±3.05)%, t1= 5.853,t2= 5.502, bothP< 0.001], and the activities of Na+-K+-ATPase, Ca2+-Mg2+-ATPase and total ATPase were increased significantly [Na+-K+-ATPase (μmol·mg-1·h-1): 8.608±0.307 vs. 6.728±0.173, 6.450±0.091,t1=9.237,P1= 0.001,t2= 11.675,P2< 0.001; Ca2+-Mg2+-ATPase (μmol·mg-1·h-1): 10.523±0.036 vs. 7.910±0.209, 8.064±0.195,t1= 9.718,P1= 0.001,t2= 11.535,P2<0.001; total ATPase (μmol·mg-1·h-1): 17.041±0.324 vs. 14.150±0.182, 13.983±0.085,t1= 16.113,t2= 17.602, bothP<0.001]. There was no statistical difference in above indexes between lentivirus control group and non-infection group.Conclusion HSP70 can maintain the PC12 cells calcium homeostasis, which may be one of the important mechanisms of anti-apoptosis.

Effect of ultraviolet B irradiation on the expression of plasma membrane calcium ATPase 3 in human lens epithelial cells / 中华实验眼科杂志

Background Plasma membrane calcium ATPase 3 (PMCA3) participates in the regulation of Ca2+ level in lens epithelial cells (LECs),which may be associated with the pathogenesis of cataract.It has been proved that ultraviolet B (UVB) is one of causing-factors of cataract.However,the effect of UVB on the expression of PMCA3 in LECs is unclear.Objective This study was to investigate the effects of UVB irradiation on the expression of PMCA3 in human LECs B-3.Methods HLE B-3 cells were cultured and passaged.The cells were exposed to 0,5,10 and 20 mJ · s/cm2 UVB for 0,20,40,80 s,respectively and further cultured for 24,48 and 72 hours.MTT assay was used to detect the cell proliferation rate.JC-1 staining was used to detect the changes of mitochondrial membrane potential (△ψm) in the cells.The intracellular reactive oxygen species (ROS) level was detected by DCFH-DA staining,and cell apoptosis was evaluated using annexin V-FITC/PI staining.In addition,the intracellular calcium ion (Ca2+) concentration in the cells was assayed with Fluo-3/AM staining.The expression levels of PMCA3 mRNA and PMCA protein in the HLE B-3 cells were detected by real-time PCR and Western blot,respectively.Results The survival rates of the cells were significantly reduced with the increase of irradiated intensity of UVB and the lapse of time (Fgroup =72.411,P =0.000 ; Ftime =36.588,P =0.000),and the survival rates of the cells in the 10 mJ · s/cm2 and 20 mJ · s/cm2 UVB for 24 hours were (75.3 ± 2.2) ％ and (48.7 ±4.5) ％,respectively,which were significantly lower than those in the 0 mJ · s/cm2 UVB group ([100.0±0.0] ％) (P=0.001,0.000).The survival rates of the cells in the 5,10,20 mJ · s/cm2 UVB for 48 hours were (84.9± 1.2) ％,(69.3±17.4)％ and (32.8±4.5)％,showing significant declines in comparison with the 0 mJ · s/cm2 UVB group ([100.0±0.0] ％) (P =0.047,0.000,0.000).In 72 hours following 5,10,20 mJ · s/cm2 UVB irradiation,the survival rates of the cells were (55.1 ± 3.0) ％,(42.1 ± 1.9) ％ and (26.1 ±4.7) ％,respectively,with significant differences in comparison with the 0 mJ · s/cm2 UVB group ([100.0 ± 0.0] ％) (P =0.000,0.000,0.000).JC-1staining exhibited the intracellular red fluorescence in the normal cells group.However,in the 5 mJ · s/cm2 UVB group,weak green fluorescence was seen,and the green fluorescence was enhanced in the 10 mJ · s/cm2 and 20 mJ · s/cm2 UVB groups.After irradiated by 5,10 and 20 mJ · s/cm2 UVB,the ROS levels in the cells increased from 0.4％ to 35.8％,51.9％ and 76.7％,respectively.The apoptosis and necrosis rate of the cells was 2.0％ in the 0 mJ · s/cm2 UVB and 4.2％,7.6％,15.1％ in the 5,10,20 mJ · s/cm2 UVB groups,respectively.The Ca2+ level raised by (1.2±0.1) and (1.3±0.1) folds in the 10 and 20 mJ · s/cm2 UVB groups more than that in the 0 mJ · s/cm2 group (P =0.039,0.004).The expression levels of PMCA3 mRNA in the cells were significantly reduced (P=0.001,0.004,0.000),and the expression levels of the PMCA protein were declined in the 5,10 and 20 mJ · s/cm2 UVB groups compared with the 0 mJ · s/cm2 UVB group (P=0.000,0.000,0.001).Conclusions UVB irradiation causes cataract probably through downregulating the expression of PMCA3 in human LECs and inducing apoptosis of LECs in a dose-and time-dependent manner.

Effect of Hypoxic Paracrine Media on Calcium-Regulatory Proteins in Infarcted Rat Myocardium

BACKGROUND AND OBJECTIVES: An increase in intracellular calcium concentration due to loss of Ca2+ homeostasis triggers arrhythmia or cardiac cell death in the heart. Paracrine factors released from stem cells have beneficial cardioprotective effects. However, the mechanism of modulation of Ca2+ homeostasis by paracrine factors in ischemic myocardium remains unclear. MATERIALS AND METHODS: We isolated rat bone marrow-derived mesenchymal stem cells (MSCs), and prepared paracrine media (PM) from MSCs under hypoxic or normoxic conditions (hypoxic PM and normoxic PM). We induced rat myocardial infarction by left anterior descending ligation for 1 hour, and treated PM into the border region of infarcted myocardium (n=6/group) to identify the alteration in calcium-regulated proteins. We isolated and stained the heart tissue with specific calcium-related antibodies after 11 days. RESULTS: The hypoxic PM treatment increased Ca2+-related proteins such as L-type Ca2+ channel, sarcoplasmic reticulum Ca2+ ATPase, Na+/K+ ATPase, and calmodulin, whereas the normoxic PM treatment increased those proteins only slightly. The sodium-calcium exchanger was significantly reduced by hypoxic PM treatment, compared to moderate suppression by the normoxic PM treatment. CONCLUSION: Our results suggest that hypoxic PM was significantly associated with the positive regulation of Ca2+ homeostasis in infarcted myocardium.

Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.