Crocin inhibits glutamate-induced apoptosis of retinal ganglion cells / 西安交通大学学报(医学版)

Objective To evaluate the effects and underlying mechanisms of crocin on glutamate-induced apoptosis of retinal ganglion cells (RGCs) by affecting extracellular calcium influx.Methods Primary rat retinal ganglion cells were isolated and stimulated with glutamate at concentrations of 0.1 mmol/L and 1 mmol/L for 24 h or 48 h,respectively,to establish apoptosis model of RGCs.Afterwards,crocin of different doses (0.1,1.0 and 3.0 μmol/L) was used to treat the glutamate-induced RGCs for 12 h;then cell apoptosis was detected by Annexin V-FITC/PI staining.The intracellular calcium concentration was determined by FIuo-3/AM fluorescent labeling.Western blot was used to examine the effect of crocin on Ca2+-mediated apoptotic signal molecules calpain and CaMKII.The mitochondrial membrane potential was detected by JC-1 staining and mitochondrial apoptosis-related signaling molecules Caspase-3,Caspase-9 and Bcl-2/Bax were evaluated by Western blot,respectively.Results In comparison with the untreated controls,the cell apoptosis of RGCs exposed to 0.1 mmol/L of glutamate for 24 h did not significantly change (P＞ 0.05).However,apoptosis rate of the cells reached (43.050 ± 2.616) ％ when the stimulation time lasted for 48 h and showed a significant increase (P＜0.01).Treatment with higher-dose glutamate (1 mmol/L) significantly increased apoptosis of RGCs at 24 h (46.450±1.061)％ and 48 h (45.500±3.253)％ compared with the controls (P＜0.01).RGCs were induced by 1 mmol/L of glutamate for 12 h,followed by the treatment with crocin at concentrations of 0.1,1.0 and 3.0 μmol/L,respectively.Each dose of crocin could significantly inhibit cell apoptosis in the dose-dependent manner (P＜0.01).In addition,crocin at 1.0 μmol/L blocked glutamate-induced extracellular calcium influx,inhibited the expression of calcium-dependent proteins Calpainl and CaMK Ⅱ.Moreover,crocin at the dose of 1.0 μmol/L also increased mitochondrial membrane potential,suppressed the expressions of Caspase-3 and Caspase-9,and elevated Bcl-2/Bax ratio.Cornclusion Crocin inhibits glutamate-induced apoptosis of retinal ganglion cells through suppressing extracellular calcium influx,thereby blocking calcium-dependent and mitochondria-dependent apoptosis signaling pathways.

Dexmendetomidine decreases spontaneous contraction of duodenal smooth muscle of rabbits in vitro / 中国药理学通报

Aim To observe the influences of dexmen-detomidine on the spontaneous contraction of duodenal smooth muscle of rabbits in vitro and explore the mech-anisms.Methods The rabbits ( male or female ) were stunned and the duodenums were isolated .The sam-ples of duodenal segments were connected with tension transducer , which were then put into oxygen saturation Krebs-Henseleit ( K-H) solution .The influences of dex-mendetomidine on amplitude ( AM ) and frequency ( FR ) of duodenal smooth muscle were recorded by BL-420 F biological signal processing system .The cu-mulative dosing method was used to observe the differ-ent concentrations of dexmedetomidine on duodenal smooth muscle spontaneous contraction .Glibenclamide ( Gli) was added to K-H solution before dexmendeto-midine.In the calcium-free K-H solution, calcium chloride and rynodine were added before dexmendeto-midine.The mechanisms of dexmendetomidine were studied .Results ① Dexmendetomidine reduced the amplitude of spontaneous contraction of duodenal smooth muscle in rabbits in a dose-dependent manner ( P0.05 ) .② Gli ( P <0.05 ) partly abolished the inhibitory effects of dexmendetomi-dine on duodenal smooth muscle .③ Dexmendetomi-dine inhibited the contraction of duodenum smooth muscle induced by calcium chloride ( P <0.05 ) and rynodine ( P<0.05 ) application into calcium-free K-H solution.Conclusion Dexmendetomidine inhibits the spontaneous contraction of duodenal smooth muscle of rabbits in vitro.The mechanisms may be related to ac-tivating ATP sensitive potassium channels , inhibition of the extracellular calcium influx via cell membrane and intracellular calcium release via sarcoplasmic reticulum in duodenal smooth muscle .

Mepivacaine-induced intracellular calcium increase appears to be mediated primarily by calcium influx in rat aorta without endothelium / 대한마취과학회지

BACKGROUND: Mepivacaine induces contraction or decreased blood flow both in vivo and in vitro. Vasoconstriction is associated with an increase in the intracellular calcium concentration ([Ca2+]i). However, the mechanism responsible for the mepivacaine-evoked [Ca2+]i increase remains to be determined. Therefore, the objective of this in vitro study was to examine the mechanism responsible for the mepivacaine-evoked [Ca2+]i increment in isolated rat aorta. METHODS: Isometric tension was measured in isolated rat aorta without endothelium. In addition, fura-2 loaded aortic muscle strips were illuminated alternately (48 Hz) at two excitation wavelengths (340 and 380 nm). The ratio of F340 to F380 (F340/F380) was regarded as an amount of [Ca2+]i. We investigated the effects of nifedipine, 2-aminoethoxydiphenylborate (2-APB), gadolinium chloride hexahydrate (Gd3+), low calcium level and Krebs solution without calcium on the mepivacaine-evoked contraction in isolated rat aorta and on the mepivacaine-evoked [Ca2+]i increment in fura-2 loaded aortic strips. We assessed the effect of verapamil on the mepivacaine-evoked [Ca2+]i increment. RESULTS: Mepivacaine produced vasoconstriction and increased [Ca2+]i. Nifedipine, 2-APB and low calcium attenuated vasoconstriction and the [Ca2+]i increase evoked by mepivacaine. Verapamil attenuated the mepivacaine-induced [Ca2+]i increment. Calcium-free solution almost abolished mepivacaine-induced contraction and strongly attenuated the mepivacaineinduced [Ca2+]i increase. Gd3+ had no effect on either vasoconstriction or the [Ca2+]i increment evoked by mepivacaine. CONCLUSIONS: The mepivacaine-evoked [Ca2+]i increment, which contributes to mepivacaine-evoked contraction, appears to be mediated mainly by calcium influx and partially by calcium released from the sarcoplasmic reticulum.

The Relaxant Effect of Propofol on Isolated Rat Intrapulmonary Arteries

Propofol is a widely used anesthetic. Many studies have shown that propofol has direct effects on blood vessels, but the precise mechanism is not fully understood. Secondary intrapulmonary artery rings from male rats were prepared and mounted in a Multi Myograph System. The following constrictors were used to induce contractions in isolated artery rings: high K+ solution (60 mmol/L); U46619 solution (100 nmol/L); 5-hydroxytryptamine (5-HT; 3 micromol/L); or phenylephrine (Phe; 1 micromol/L). The relaxation effects of propofol were tested on high K+ or U46619 precontracted rings. Propofol also was added to induce relaxation of rings preconstricted by U46619 after pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). The effects of propofol on Ca2+ influx via the L-type Ca2+ channels were evaluated by examining contraction-dependent responses to CaCl2 in the absence or presence of propofol (10 to 300 micromol/L). High K+ solution and U46619 induced remarkable contractions of the rings, whereas contractions induced by 5-HT and Phe were weak. Propofol induced dose-dependent relaxation of artery rings precontracted by the high K+ solution. Propofol also induced relaxation of rings precontracted by U46619 in an endothelium-independent way. Propofol at different concentrations significantly inhibited the Ca2+-induced contractions of pulmonary rings exposed to high K+-containing and Ca2+-free solution in a dose-dependent manner. Propofol relaxed vessels precontracted by the high K+ solution and U46619 in an endothelium-independent way. The mechanism for this effect may involve inhibition of calcium influx through voltage-operated calcium channels (VOCCs) and receptor-operated calcium channels (ROCCs).

Role of calcium channels responsible for phenylephrine-induced contraction in rat aorta 3 days after acute myocardial infarction / 대한마취과학회지

BACKGROUND: Phenylephrine (PE) produces tonic contraction through involvement of various calcium channels such as store-operated calcium channels (SOCCs) and voltage-operated calcium channels (VOCCs). However, the relative contribution of each calcium channel to PE-induced contraction has not been investigated in isolated rat aorta of early acute myocardial infarction (AMI). METHODS: Endothelium-denuded rat aortic rings from rats 3 days after AMI or sham-operated (SHAM) rats were prepared in an organ chamber with Krebs-Ringer bicarbonate solution for isometric tension recording. We assessed the PE dose-response relationships in 2.5 mM calcium medium for both groups. The same procedure was repeated using rings pretreated with the SOCC inhibitor 2-aminoethoxydiphenyl borate, sarco/endoplasmic-reticulum calcium ATPase inhibitor thapsigargin (TG), diacyl glycerol lipase inhibitor RHC80267, and sodium-calcium exchanger inhibitor 3,4-dichlorobenzamil hydrochloride for 30 minutes before addition of calcium. When ongoing tonic contraction was sustained, dose-response curves to the VOCC inhibitor nifedipine were obtained to assess the relative contribution of each calcium channel under various conditions. RESULTS: The effect of SOCC induction with TG pretreatment on PE-induced contraction was significantly lower in the AMI group compared to the SHAM group. In addition, there were significant decreases in the sensitivity and efficacy of the VOCC inhibitor nifedipine on PE-induced contraction in the AMI group. CONCLUSIONS: Results suggest that the change of vascular reactivity of PE in rat aorta 3 days after AMI is characterized by a decreased contribution of L-type VOCCs. The enhanced VOCC-independent calcium entry mechanisms after AMI can be mediated by enhanced capacitative calcium entry through the activation of SOCCs.

Calcium-influx response and ERK phosphorylation in human microglia triggered by HIV-1 glycoprotein gp120 / 中国病理生理杂志

AIM: The aim of the present study is to explore the effects and mechanisms of HIV-1 glycoprotein gp120 on calcium-influx and ERK-phosphorylation of human microglia.METHODS: The level of intracellular calcium of human microglia grown on coverslip,which was loaded by calcium-probe,Fluo-4,and then treated in various experimental processing,was detected by confocal microscopy with time resolution mode.The binding of gp120 to human microglia was determined with confocal microscopy or flow cytometry after treatment with gp120 and stained with anti-gp120-FITC antibody.Phosphorylation of ERK within human microglia with or without gp120 stimulation was analyzed with confocal microscopy following the direct immuno-staining with anti-phosphorylated ERK antibody.RESULTS: The results of confocal microscopy showed that calcium-influx response was triggered by HIV-1 glycoprotein gp120 in human microglia.The results from analysis by confocal microscopy and flow cytometry showed that gp120 was able to bind to human microglia.ERK phosphorylation was enhanced in human microglia stimulated with gp120.CONCLUSION: HIV-1 glycoprotein gp120 induces calcium-influx in human microglia and enhances ERK phosphorylation in human microglia,indicating that gp120 is an activator of human microglia.So gp120 may be involved in the pathogenesis of HIV-associated dementia.

Effects of parathyroid hormone on calcium transsacolemma in cultured heart cells / 中国药理学通报

In order to obtain some evidences of the role of parathyroid hormone (PTH) in regulating calcium metabolism, and explore the mechanisms by which PTH regulates calcium in the heart, 45Ca was used to study the influence of bovine PTH1-34 fragment (bPTH1-34) on calcium transsacolemma. The results indicate that bPTH1-34(l?I0-7mol ? L-1) increases beating rate of heart cells under normal state and it markedly increases the rapid (5 min) and the slow (120 min) phases of 45Ca influx in heartcells (under normal, KC1 10-5 mol?L-1 high K+depolarised, NA 10-7mol?L-1receptors activated states), but it could also reduce 45Ca efflux from the cultured heart cells. It suggests that the mechanisms caused by bPTH1-34 which enhances the cytosolic calcium concentration and myocardial beating rate may be related with cAMP level elevated in heart cells.

EFFECT OF TAURINE ON MODULATING CALCIUM OF CARDIAC MUSCLES IN RATS / 中国药理学通报

The effect of Taurine ( Tau)on 45Ca influx of isolated left ventricular muscles in rats has been investigated. The 45Ca influx of the preparations untreated with Tau, are affected by the concentrations of calcium in KH solution. when the concentration of calcium is 0.62; 1.25; and 1.87mmol/L respectively, the 45Ca influx is 1.02? 0.25; 1.37?0.14 and 1.45?0.14 ?mol/g wet tissue, respectively. After adding Tau, the situations above were changed significantly. It was shown that is 1.11 ? 0.11, 1.45?0.12, and 1.48 ? 0.09 ?mol/g wet tissue in low Ca2?; 1.19?0.07, 1.14 ? 0.23, and 0.97?0.24 ?mol/g wet tissue in normal Ca2?; and 1.12?0.05,0.58?0.18 and 0.53?0.10 ?mol/g wet tissue in high Ca2? treated with Tau 10, 20, and 40 mmol/L, respectively. The results show that the varied ,Ca2? concentrations in KH solution can affect 45Ca influx of cardiac muscles, but Taurine has a bisphasic modulation o,f calcium influx in the same tissue. The effects of modulating calcium may play an important role for anti-arrhythmic mechanism of Taurine.

Effects of Berberine on Cytosolic cAMP and Calcium Influx of Rabbit Platelets / 第二军医大学学报

We found that the platelet aggregation rates induced by ADP, arachidonic acid (AA), collagen and A23187 had an apparent decreasing after injection of berberine into the vein of rabbits, and also found that berberine had an apparent inhibition of calcium influx into platelet induced by A23187 at lower concentrations. We demonstrated that berberine had no obvious effect on cAMP level in the intact platelet of rabbits.

EFFECT OF HOMOCYSTEINE ON POTENTIATING ?ETA-AMYLOID NEUROTOXICITY / 营养学报

Objective To examine the possibility that homocysteine(Hcy) may augment ?-amyloid(A?) neurotoxicity.Method Cultured hippocampal neurons were treatd with Hcy and/or A?42 and it's apoptosis,calcium influx,DNA damage and oxidative injury were examined.Results Combined treatment with 250?mol/L Hcy and 10?mol/L A?42 increased the apoptosis rate of hippocampal neurons significantly more than either agent alone,and even the sum of each agent treated alone.The combined treatment significantly exceeded the cytosolic calcium accumulation and MDA obtained with either alone or the sum of each agent.A significant increase in DNA damage also occurred in neurons exposed to A?42 in the presence of Hcy,but the magnitude of the increase was not greater than that seen with either treatment alone.Conclusion Hcy could potentiate A? neurotoxicity by neuron exitotoxicity,oxidative stress and inducing apoptosis.