Hydraulic retention time on vinasse stabilisation with limestone in the acidogenic phase of anaerobic digestion / Tempo de retenção hidráulica em vinhaça estabilizada com calcário na fase acidogênica da digestão anaeróbia

ABSTRACT: The main problem in anaerobic digestion of low-protein residues is the instability caused acidity. The use of limestone at the same time as a neutralizing agent and support material is innovative because stones wear allows the slow release of the calcium carbonate thereby eliminating dispersers. Free calcium content in the system was measured in two plug flow reactors filled with vinasse at initial pH of 4.50. The proportion of 1.8 tonnes of limestone per m³ of vinasse was evaluated at the Hydraulic Retention Times (HRT) of 24, 48, 72, 96 and 120 hours, allowing stabilisation at 96 hours. The ratio of Volatile Acids/Total Alkalinity (VA/TA) ranged from 0.2 to 0.4 and the pH reached 7.0, at the HRT of 120 hours. Increasing the HRT also increased the volatile total solids (VTS) and fixed total solids (TFS) in a similar profile to the measured free calcium content, but calcium remained at the appropriate level of 100 to 250mg l-1. The proportion of limestone/vinasse was adequate to ensure stabilisation, but it is not recommended to reduce the HRT below 96 hours due to the risk of compromising the stability of the anaerobic system.

RESUMO: O maior problema na digestão anaeróbia de resíduos de baixo teor de proteína é a instabilidade por acidez. O uso de calcário ao mesmo tempo como agente neutralizador e material suporte é inovador porque permite eliminar o dosador pois o desgaste das pedras permite a liberação lenta do carbonato de cálcio. O teor de cálcio livre no sistema foi medido em dois reatores tipo Plug flow, preenchidos com vinhaça com pH inicial de 4,50. A proporção de 1,8 toneladas de calcário por m³ de vinhaça foi avaliada com os Tempos de Retenção Hidráulica (TRH) de 24, 48, 72, 96 e 120 horas, permitindo a estabilização em 96 horas. Com um TRH de 120 horas, a relação de Ácidos Voláteis/Alcalinidade (AV/AC) variou de 0,2 à 0,4 e o pH atingiu 7,0. Aumentando o TRH também aumentaram os sólidos voláteis totais (SVT) e sólidos totais fixos (STF) em um perfil semelhante ao teor de cálcio livre medido, mas mantendo níveis adequados de 100 a 250mg L-1. A proporção de calcário/vinhaça mostrou-se adequada para garantir a estabilização, mas não se recomenda a redução do TRH abaixo de 96 horas devido ao risco de comprometer a estabilidade do sistema anaeróbio.

Identification of Lys49-PLA2 from crude venom of Crotalus atrox as a human neutrophil-calcium modulating protein

We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca2+]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca2+ infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 microM) and SKF-96365 (20 microM) signifi cantly inhibited K49-PLA2-induced [Ca2+]i increase. These results suggest that K49-PLA2 modulates [Ca2+]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.

N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca²⁺ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca²⁺]ᵢ) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca²⁺]ᵢ in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca²⁺]ᵢ in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca²⁺]ᵢ in human neutrophils was observed. In Ca²⁺-free buffer, NAC- and cysteine-induced [Ca²⁺]ᵢ increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca²⁺]ᵢ in human neutrophils occur through Ca²⁺ influx. NAC- and cysteine-induced [Ca²⁺]ᵢ increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na⁺-free HEPES, both NAC and cysteine induced a marked increase in [Ca²⁺]ᵢ in human neutrophils, arguing against the possibility that Na⁺-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca²⁺]ᵢ increasing activity. Our results show that NAC and cysteine induce [Ca²⁺]ᵢ increase through Ca²⁺ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

TRPV1 in Salivary Gland Epithelial Cells Is Not Involved in Salivary Secretion via Transcellular Pathway

Transient receptor potential vanilloid subtype 1 (TRPV1) was originally found in sensory neurons. Recently, it has been reported that TRPV1 is expressed in salivary gland epithelial cells (SGEC). However, the physiological role of TRPV1 in salivary secretion remains to be elucidated. We found that TRPV1 is expressed in mouse and human submandibular glands (SMG) and HSG cells, originated from human submandibular gland ducts at both mRNA and protein levels. However, capsaicin (CAP), TRPV1 agonist, had little effect on intracellular free calcium concentration ([Ca2+]i) in these cells, although carbachol consistently increased [Ca2+]i. Exposure of cells to high temperature (>43degrees C) or acidic bath solution (pH5.4) did not increase [Ca2+]i, either. We further examined the role of TRPV1 in salivary secretion using TRPV1 knock-out mice. There was no significant difference in the pilocarpine (PILO)-induced salivary flow rate between wild-type and TRPV1 knock-out mice. Saliva flow rate also showed insignificant change in the mice treated with PILO plus CAP compared with that in mice treated with PILO alone. Taken together, our results suggest that although TRPV1 is expressed in SGEC, it appears not to play any direct roles in saliva secretion via transcellular pathway.

Cloning and expression of rotavirus nonstructural protein 4 and its interference effect on intrancellu-lar calcium homeostasis / 中华微生物学和免疫学杂志

Objective To construct prokaryotic expression systems of genes of intact rotavirus non -structural protein 4 (NSP4) and 86 to 175 amino acid peptide of NSP4 (NSP486-175) and to identify the bio-activities of the recombinant expression products .Methods Colloidal gold assay was used to screen group A rotavirus antibody-positive stools , from which the viral RNA was extracted and transcribed into cDNA .Then NSP4 gene and NSP486-175 gene were amplified by PCR and inserted into pET-28a(+) vector to construct the prokaryotic expression plasmids of pET-28a(+)-NSP4 and pET-28a(+)-NSP486-175 , which were transformed into E.coli BL21( DE3) and inducted by IPTG .The expressed NSP486-175 protein was purified by Ni-NTA af-finity chromatography and examined by Western blot .Fluo-3 AM ester was used to label intracellular free Ca2+( [ Ca2+] i) in Caco-2 cells and laser scanning confocal microscopy was performed to detect the effect of NSP486-175 protein on [Ca2+]i.Results DNA sequencing indicated that NSP4 gene and NSP486-175 gene were consistent with the corresponding sequences in NCBI .The intact NSP4 had difficulty in expressing in E.coli. NSP486-175 protein mainly existed in soluble form with a relative molecular weight of 10×103 .Fluorescence in-tensity and distribution were changed by exogenous addition of NSP 486-175 to cultured Caco-2 cells.Conclu-sion The expression vectors of pET-28a(+)-NSP4 and pET-28a(+)-NSP486-175 were successfully construc-ted and the target protein could induce [Ca2+]i imbalance.This phenomenon preliminarily indicates that the prokaryotic expressed NSP486-175 protein has biological activity and can be further used to elucidate rotavirus pathogenic mechanisms and perform vaccine research .

Effect of electroacupuncture on brain cells apoptosis and nerve function recovery after focal cerebral ischemia-reperfusion in rats / 中华行为医学与脑科学杂志

Objective To investigate the influence of acpuncture on free calcium in rat brain cells after focal cerebral ischemia reperfusion.Methods 145 adult male SD rats were randomly divided into control group,simple ischemia reperfusion group and acupuncture with ischemia reperfusion group.The middle cerebral artery occlusion/reperfusion (MCAO/R) rat model was established by the modified Longa occlusion method. ①The part of free calcium in rat brain cells,focal cevebral ischemia model of rats were made by thread locking up the blood vessel for 15 min.30 min later after reperfusion, the Baihui and Shuigou Point in Du meridian were acupunctured electrically 30 min.After 3h, 6h and 12h, the rat was killed and its brain cells were made into single cell suspension,marked by Fluo-3/AM.The fluorescence optical density was recorded by laser confocal microscopy.②The part of nerve functional reconstruction, focal cevebral ischemia model of rats were made by thread locking up the blood vessel for 12 hours.30 min later after reperfusion, the Baihui and Shuigou Point in Du meridian were acupunctured electrically 30 min.After 7 d, 14 d,30 d,60 d and 90 d, the rat was forced to detect it's strength of the dog.Results ①Free calcium in rats of acupuncture therapy group(6h:10.96±1.18;2h:20.9±4.37) was significantly less than that in control group in 6 h and 12 h after reperfusion (6 h: 16.87 ± 3.56,12 h: 34.10 ±1.06)(P<0.05).②The dog in rats of acupuncture therapy group was significantly more than that in control group in 7 d, 14 d after reperfusion (P＜ 0.05 ).No difference of the dog was detected in 30 d ,60 d and 90 d after reperfusion between the two groups.Conclusion Acupunture could decreases the concentration of free calcium and the expression of Caspase-3 mRNA in rat brain cells after focal cerebral ischemia reperfusion, and it can facilitate the recovery of nerve function.

Action mechanism of hyperin on neonatal rat's neuron with anoxia-reoxygenation / 中国药理学通报

Aim To study the action mechanism of hyperin(Hyp) on neonatal rat's neuron with anoxia/reoxygenation(A/R).Methods The dissociated neonatal rat brain cells were subjected to 30 min of anoxia or followed 40 min of reoxygenation.Lactate dehydrogenase(LDH),malondialdehyde(MDA)and nitric oxide(NO)in the supernatant were measured.The intracellular free calcium concentration([Ca~(2+)]_i)in brain cells was assayed with Fura 2-AM method.Results Anoxia induced a significant increase of LDH in the supernatant from (62.0±13.0) U·L~(-1)(Sham group)to (116.0±16.6) U·L~(-1)(Control group,P<0.01),and reoxygenation markedly increased LDH and MDA in the supernatant from (45.6±9.2) U·L~(-1) and (9.1±0.9) μmol·L~(-1)(Sham group)to (106.0±17.4) U·L~(-1) and (16.4±2.7) μmol·L~(-1)(Control group,P<0.01),respectively.In the range of 1.0 ～ 16.0 μmol·L~(-1),Hyp markedly and concentration-dependently inhibited anoxia-or reoxygenation-evoked increases of LDH and MDA.1.0～16.0 μmol·L~(-1) Hyp not only inhibited anoxia-induced increase of NO in the supernatant and rise of [Ca~(2+)]_i in brain cells(P<0.05 or P<0.01),but also attenuated reoxygenation-evoked increases of NO and[Ca~(2+)]_i(P<0.05 or P<0.01),Hyp 16.0 μmol·L~(-1) significantly reduced NO and[Ca~(2+)]_i from (34.4±6.3) μmol·L~(-1) and (640±94) nmol·L~(-1) to (25.0±5.1) μmol·L~(-1) and (331±56) nmol·L~(-1),respectively.Conclusion The protective effect of Hyp on A/R-injured neurons may be related to the inhibition of overload of[Ca~(2+)]_i,NO release and lipid peroxidation.

Effect of Emodin on intracellular calcium concentration ([Ca~(2+)]i) and apoptosis of hepatic cells after simulated cold ischemia-reperfusion / 西安交通大学学报(医学版)

Objective To investigate the effect of Emodin on intracellular calcium concentration ([Ca~(2+)]i) and apoptosis of hepatic cells after simulated cold ischemia-reperfusion. Methods Glucose-oxygen deprivation, low temperature, subsequent reoxygenation and rewarming were used to induce ischemia-reperfusion injury model in cultured hepatic cells which were divided into 4 groups: control group and Emodin-treated group(100, 10 and apoptosis rate were determined by flow cytometry (FCM) respectively; the content of lactate dehydrogenase (LDH) in supernatant was tested. Results Intracellular calcium fluorescence intensity in Emodin-treated groups of high, medium and low density was 24.12±0.51, 26.35±1.34 and 39.12±1.94, respectively, which were significantly lower than 105.29±1.01 in control group(P<0.01). Apoptosis rate in Emodin-treated groups of high, (179.67±18.57)u/L in Emodin-treated groups of medium and high density respectively, which were significantly lower than (351.33±34.16)u/L in control group(P<0.01). Conclusion Emodin could reduce [Ca~(2+)]i and inhibit apoptosis of hepatic cells after simulated cold ischemia-reperfusion, thus protecting hepatic cells effectively.

Effects of aspirin on [Ca~(2+)] i and NGB in cultured neural cells of newborn rat under chemical-induced hypoxic exposure / 基础医学与临床

Objective To investigate the protective effects of aspirin on hypoxic brain neural cells of rat and the effects of extracellular calcium on the neuroprotective effect of aspirin. Methods The hypoxic cell model was established by adding Na_2S_2O_4 to the calcium or calcium free medium. Cultured primary cortical neurons of rat were pretreated with ASA in vitro. The change of intracellular free calcium and neuroglobin (NGB) were observed and analyzed by laser scanning confocal microscope. Results The expression level of [Ca~(2+)] i and NGB increased significantly in the chemical hypoxia group ( P < 0. 05 ). ASA can attenuate the increase of [ Ca~(2+) ] i and NGB in the hypoxic group and calcium-free hypoxia group(P <0. 05). Conclusion Aspirin can inhibit calcium overload and the hypoxia-induced expression of NGB, so protect rat brain cells against hypoxia.

Apoptosis of HepG2 Cell Induced by Ouabain and Cinobufogenin through ERK Signaling Pathway / 中国药房

OBJECTIVE:To investigate the effect of ouabain and cinobufogenin on the apoptosis of HepG2 cell in order to study its apoptosis mechanism through ERK signaling pathway. METHODS:The activity of Na+/K+-ATPase was detected with HepG2 cell as target. The morphological changes of HepG2 cell and DNA injury were determined by Hoechst 33342 fluorescent staining and single cell gel electrophoresis (SCGE) respectively. The changes of the concentration of calcium ion were investigated by Fura-3 AM, and the expressions of Caspase-3 and ERK were detected by Western blotting assay. RESULTS:Ouabain and cinobufogenin inhibited the activity of Na+/K+-ATPase in HepG2 cell. Hoechst 33342 fluorescent staining showed that the clear apoptotic characteristics of HepG2 cell. The results of SCGE demonstrated that DNA double-strand of HepG2 cell was injured by ouabain and cinobufogenin. The concentration of calcium ion also increased after the treatment of ouabain and cinobufogenin according to the result of Fura-3 AM assay. The results of Western blot proved that ouabain and cinobufogenin promoted the production of p-ERK and the activition of caspase-3. CONCLUSION:Ouabain and cinobufogenin can induce the apoptosis of HepG2 cell and DNA damage, the increase of free calcium concentration and the activity of Caspase-3. ERK signaling pathway plays an important role in the apoptosis of HepG2 induced by ouabain and cinobufogenin.

Suplementación oral de calcio en adolescentes embarazadas / Calcium oral supplementation in adolescent pregnant women

Objetivo: Determinar el efecto de la administración oral de calcio en adolescentes embarazadas de bajo nivel socioeconómico sobre las concentraciones de calcio ionizado plasmático y libre intracelular. Métodos: En un ensayo clínico controlado doble-ciego aleatorizado se estudiaron 52 mujeres, 26 (50%) adolescentes embarazadas que recibieron 600 mg de calcio elemental y 26 (50%) adolescentes embarazadas que recibieron 600 mg de placebo entre las semanas 17 y 19 de embarazo. Los niveles pre-tratamiento y post-tratamiento de calcio ionizado plasmático y libre intracelular se evaluaron en ambos grupos de acuerdo con la intención de tratamiento. Resultados: Se analizaron 48 adolescentes embarazadas que completaron el estudio (24 en el grupo de calcio y 24 en el grupo de placebo). Las características sociodemográficas de los grupos fueron comparables (p=0.92) al igual que la ingesta basal de calcio en su dieta (p=0.62). La suplementación oral de calcio por intención de tratamiento no modificó las concentraciones de calcio ionizado plasmático (1.19+0.04 mmol/l vs. 1.23+0.02 mmol/l, p=0.56) ni las concentraciones del calcio ionizado libre intracelular (116.2 mmol/l vs. 89.7 mmol/l, p= 0.91), se observó un resultado semejante en las embarazadas que recibieron placebo (1.20+0.05 mmol/l vs. 1.19+0.03 mmol/l p=0.86; 116.2 mmol/l vs. 137.5 mmol/l, p=0.16, respectivamente). Conclusiones: La administración oral de calcio en adolescentes embarazadas de bajo nivel socioeconómico no modificó ni las concentraciones plasmáticas ni las intracelulares del calcio ionizado lo que podría explicar en parte el poco efecto preventivo del uso del calcio como única medida de intervención para prevenir la preeclampsia.

Objective: To determine the effect of oral administration of calcium on plasma and ionized free calcium concentration in healthy adolescent pregnant women. Methods: In a double blind randomized controlled clinical trial were recruited 48 healthy adolescent pregnant women, 24 (50%) received 600 mg of elemental calcium and 24 (50%) received 600 mg of lactose placebo. At the inclusion time the plasma and intracellular free calcium concentrations were measured by standardized techniques. One month later the plasma and intracellular free calcium concentrations in both groups were measured. Results: At the inclusion time and one month after treatment both groups were comparable for sociodemographic characteristics and the basal intake of calcium (p=0.92, p=0.62). Calcium supplementation did not modify the concentrations of plasma ionized calcium (1.19+0.04 mmol/l vs. 1.23+0.02 mmol/l, p=0.56) and the free intracellular calcium concentration (mmol/l vs. 89.7 mmol/l, p=0.91); similar effects were observed with the placebo treatment (1.20+0.05 mmol/l vs. 1.19+0.03 mmol/l p=0.86; 116.2 mmol/l vs 137.5 mmol/l, p=0.16, respectively). Conclusions: Oral administration of 600 mg of elemental calcium in adolescent pregnant women did not induce changes in the plasma and intracellular ionized free calcium concentrations and could explain in part the lack effect of this only supplementation in preeclampsia prevention.

Effects of aspirin on [Ca~(2+)]i and NGB in cultured neural cells of newborn rat under chemical-induced hypoxic exposure / 基础医学与临床

Objective To investigate the protective effects of aspirin on hypoxic brain neural cells of rat and the effects of extracellular calcium on the neuroprotective effect of aspirin. Methods The hypoxic cell model was established by adding Na2S2O4 to the calcium or calcium free medium. Cultured primary cortical neurons of rat were pretreated with ASA in vitro. The change of intracellular free calcium and neuroglobin (NGB) were observed and analyzed by laser scanning confocal microscope. Results The expression level of [Ca2+]i and NGB increased significantly in the chemical hypoxia group(P

Effect and Mechanism of Epidermal Growth Factor on Proliferation of GL15 Gliomas Cell Line / 华中科技大学学报（医学）（英德文版）

The effects of epidermal growth factor (EGF) on proliferation of G 15 glioma cells and the possible mechanisms were investigated. GFAP and EGFR expression was detected by immunohistochemical method. After the cells were treated with EGF at different concentrations, cell count method was used to determine the proliferation of glioma cells, cell cycle and apoptosis were analyzed by flow cytometry (FCM), and laser scan confocal microscope (LSCM) was used to measure the cytoplasmic free calcium. The results showed that GFAP was diffusedly expressed in GL15 cells and EGFR was over-expressed. EGF at doses of ≤ 1 ng/mL could significantly stimulate cell proliferation, cells in phase G0/G1 decreased, and those in phase S increased. EGF at doses of 10 and 100ng/ml could inhibit the cell proliferation significantly, and the apoptosis ratio in high dose of EGF group was higher than in control group. EGF could significantly induce a quick rise of intracellular free calcium, but the peak value of intracellular free calcium activated by high dose of EGF was higher than by low dose of EGF. It was suggested that EGF had a dual effect on gliomas: low dose of EGF could stimulate the cell proliferation of gliomas, but high dose of EGF could induce the cell apoptosis and inhibit the proliferation of gliomas, which might be contributed to the difference of intracellular free calcium.

The effect of Shihusan on intracellular free calcium of human retinal neural cells / 眼科

Objective To explore the effects of Shihusan on the intracellular free calcium of human retina cells and the protection from the damage of glutamate. To investigate its function and mechanism in retinal degeneration diseases, and to offer the basis on which to treat the similar disease clinically. Design Experimental study. Participants Retinal cells from normal human eye. Methods Cultivation of human retinal neural cells was perfonned. The changes of fluorescent density of intracellular free calcium of cultured cells labeled with Fluo3/AM before and after adding Shihusan and verapamil were detected with laser scanning confocal microscopy. In addition, the effect of Shihusan on calcium overload provoked by glutamic acid was observed. Main Outcome Measures The level of intracellular free calcium of retinal cells. Results The retinal cells adhered, spread and grew well in vitro. The fluorescence intensity increased 88% in glutamic acid group, decreased 14.8% and 57.3% in Shihusan group and verapamil group respectively. Shihusan couldn't decrease the intracellular Ca2+ that was increased by glutamic acid, but could inhibit the increasing tendency induced by glutamic acid. The difference was significant compared to control group (P=0.000). Conclusions Shihusan might decrease the level of intracellular free calcium in cultured human retinal cells, and resist the damage of glutamate. Its mechanism was related possibly to resisting calcium overload and inhibiting apoptosis.

Effect of Extracts from Safflower Seeds on Osteoblastic Differentiation and Intracellular Free Calcium Concentration in MC3T3-E1 Cells

Very little research has been carried out on safflower seed for the prevention and treatment of the bone deficiency diseases, including osteoporosis, which are supported by scientific evidences. In the present study, 3microliter of 0.1% dried crude extract or 2microliter of 0.1% dried aqueous fraction were shown to significantly accelerate the rate of differentiation of osteoblast. Also, the crude extract and aqueous fraction increased the [Ca2+]i of the cultured osteoblast cells: 3microliter of 0.1% dried crude extract and 2microliter of 0.1% dried aqueous fraction significantly increased the [Ca2+]i of the cultured osteoblast cells (8x104) to the extent that it deserves a considerable attention. Furthermore, the crude extract and aqueous fraction increased the [Ca2+]i of the cultured osteoblast cells, and 300microM Cd2+, specific calcium channel blocker, completely blocked the increase. Therefore, the increased [Ca2+]i of the cultured osteoblast cells by safflower seed component continued to activate calcium channel.

Comparison of ANG II-mediated Ca2+i, IP3 Production and ATPi in Isolated Renal Proximal Convoluted Tubules of Adult SHR and WKY / 대한신장학회잡지

BACKGROUND: The renin angiotensin syaimstem plays an important role in hypertension. Therefore, the purpose of this study was to investigate the comparison of responsiveness to angiotensin II (ANG II) in isolated renal proximal convoluted tubules of spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats. METHODS: Intracellualr calcium concentration ([Ca2+i) was measured using Fura- 2/AM, inositol trisphosphate (IP3) accumulation was determined by radioimmuno assay and cellular ATP content measured using the microchemilunescene method in renal proximal tubule suspension or isolated renal proximal tubules. RESULTS: When measured the ANG II-induced [Ca2+i, the young rats showed a greater response to ANG II than adult rats in both strains. The ANG II (10-7 M)-induced [Ca2+i transient in the cortical tubule suspension from adult SHR was significantly lower than that in age-matched WKY. In isolated proximal tubule segments, ANG II-induced [Ca2+i increment was only observed in S1 segments. Comparing responsiveness to ANG II in SHR and WKY, similar phenomenon was observed as experiment using tubule suspension. IP3 accumulation by ANG II also attenuated in adult SHR. The 20-minutes incubation without any exogenous substrate in proximal convoluted tubule (S1) significantly decreased cellular ATP content and ANG II (10-7 M) inhibited decrement of cellular ATP level. The effect of ANG II on cellular ATP restoration was disappeared by the treatment with losartan. CONCLUSION: From these results, the responsiveness of ANG II to AT1A receptor is attenuated in the proximal convoluted tubules of adult SHR comparing the age- mached WKY.

The Role of ATP on Reactive Oxygen Species Generation and Cellular Proliferation in BV-2 Cells / 대한소아신경학회지

PURPOSE: Adenosine triphosphate (ATP) evokes several cellular responses in microglia including propagation. However, the role of the purinoceptor on ROS generation in microglia is unclear. In order to determine the action of the purinoceptor in microglia, the effects of ATP on ROS generation and cellular proliferation in BV-2 murine microglial cells were evaluated. An additional aim of this study was to investigate signal transduction pathways using several inhibitors. METHODS: The [Ca2+] was measured using Ca2+ sensitive indicator, Fura-2/AM. ROS production was observed by fluorescence-confocal microscope and cell proliferation was evaluated by counting cell number. RESULTS: ATP increased the intracellular calcium levels ([Ca2+]i) in BV-2 cells in a dose-dependent manner. This increase was attenuated by pretreatment with a calcium chelator (EGTA) and a phospholipase C (PLC) inhibitor (U-73122) while the protein tyrosine kinase (PTK) inhibitor (genistein) had no inhibitory effects. To identify the effects of the nucleotides, ROS generation was observed in the nucleotide-stimulated BV-2 cells. The treatment with 100 M ATP induced ROS generation, but 100 M adenosine and 100 M UTP did not. To investigate the signal transduction pathway in ATP-induced ROS generation, several inhibitors were pretreated before adding ATP. ATP- induced ROS production was blocked by pretreatment with either 0.5 mM EGTA or 10 M U73122 while 40 M genistein had an inhibitory effect on ATP action. Correspondingly, 40 M KN62 (CaM kinase II inhibitor), 1 M sphingosine (protein kinase C inhibitor), 1 nM DPI (NADPH oxidase inhibitor) and 50 M mepacrine (phospholipase A2 inhibitor) could suppress ATP-induced ROS generation. The effects of ATP on cell proliferation was observed 3 days after ATP treatment and its peak velocity after 4 days. NF-kB activation was observed after the cells were incubated with 0.1 mM ATP. The maximal level of NF-kB activation was obtained with 0.3 mM ATP while higher concentrations were less effective. CONCLUSION: Overall, we conclude that ATP in BV-2 cells induces ROS generation and cell propagation. The signal transduction pathways including calcium, CaM kinase II, PLC, protein kinase C, phospholipase A2 and NADPH oxidase are involved in ATP-induced ROS generation.

Effects of sodium magnesium fructose diphosphate on free calcium concentration and nitric oxide synthase activity of ischemic synaptosome / 药学学报

Aim To study the effects of sodium magnesium fructose diphosphate (SMFD) on free calcium concentration and nitric oxide synthase activity of ischemic synaptosome, so as to explore the protective mechanisms of SMFD on cerebral ischemia. Methods The synaptosomes from normal rat brain were prepared by phase partition and cultured with oxygen-glucose deprivation to establish ischemic synaptosome model. The intrasynaptosomal free calcium concentration and nitric oxide synthase activity were detected separtately after the synaptosomes were co-incubated with SMFD (1.3 mmol*L-1) or fructose-1,6-diphosphate (FDP, 4.0 mmol*L-1) for 60 min. Results SMFD decreased the free calcium concentration and reduced the activity of nitric oxide synthase (NOS) of ischemic synaptosomes. Its effects were more powerful than those of FDP. Conclusion SMFD may protect neurons from ischemic injury by preventing intracellular Ca2+ overload and inhibiting the activity of nitric oxide synthase.

Effects of YMⅢ on vascular contractive activities and the underlying mechanism / 中国药理学通报

Aim To investigate the effects of YMⅢ,a derivative of naftopidil, on the vascular contractive activities in rabbit aorta and to explore its vasodilative mechanisms.Methods The isotonic contractions of the thoracic aorta strips from rabbits were recorded, and the effects of YMⅢ on the concentration-response curves of noradrenaline(NA),high potassium and 5-hydroxytryptamine(5-HT)were observed.Intracellular free Ca2+([Ca2+]i)was investigated in the pressence of and the absence of YMⅢ in different conditions.Results YMⅢ(10-8,5?10-8,10-7 mol?L-1)shifted the concentration-response curve of NA with a parallel manner to right, the maximum response was unchanged and the pA2 value was 8.00; YMⅢ (10-5 mol?L-1)also shifted the concentration-response curve induced by high potassium to right but with non-parallel manner,the response was depressed and the pD′2 value was 4.26. However, YMⅢ(10-7,10-6,10-5 mol?L-1)had no statistical influence on the concentration-response curve induced by 5-HT, although it tended to depress the response of the curve at 10-5 mol?L-1.In Ca2+-free medium,YMⅢ (10-8,5?10-8 and 10-7mol?L-1) significantly inhibited the transient contraction induced by NA and the long-lasting one induced by addition of Ca2+ with a concentration-dependent manner.But even at 10-5 mol?L-1,it did not inhibit the contraction induced by caffeine.Conclusions YMⅢ may be ?-adrenergic receptor blocker.Its vasodilative mechanism may be related to:blocking ?-adrenergic receptor on cell membrane resulting in the inhibition on the influx of extracellular Ca2+ and the release of intracellular calcium.

Effects of smoking and glonoine on intracellular Ca~(2+) in vascular smooth muscle cell in rabbits with atherosclerosis / 中国药理学通报

Aim To study the effects of smoking and glonoine on intracellular free Ca2+ concentration([Ca2+]i) in vascular smooth muscle cell in rabbits with atherosclerosis,and to explore the effects of smoking on atherosclerosis and biologic action of glonoine.Methods An atherosclerosis in rabbits was produced.The vascular smooth muscle cells were isolated.The cells were loaded by Fluo-3/AM.[Ca2+]i in vascular smooth muscle cell was measured by flow cytometer(FCM).The spatial distribution and the dynamic changes of [Ca2+]i in single vascular smooth muscle cells were determined by laser scanning confocal microscopy(LSCM).Results Atherosclerosis plaques in arteriae aorta were observed and the degrees were different in various groups.[Ca2+]i in vascular smooth muscle cells in rabbits with atherosclerosis markedly increased[(48.45?5.31) vs that in saline control(38.09?2.57),P