Methodology for Cross-Talk Elimination in Simultaneous Voltage and Calcium Optical Mapping Measurements With Semasbestic Wavelengths.

Most cardiac arrhythmias at the whole heart level result from alteration of cell membrane ionic channels and intracellular calcium concentration ([Ca2+] i ) cycling with emerging spatiotemporal behavior through tissue-level coupling. For example, dynamically induced spatial dispersion of action potential duration, QT prolongation, and alternans are clinical markers for arrhythmia susceptibility in regular and heart-failure patients that originate due to changes of the transmembrane voltage (V m) and [Ca2+] i . We present an optical-mapping methodology that permits simultaneous measurements of the V m - [Ca2+] i signals using a single-camera without cross-talk, allowing quantitative characterization of favorable/adverse cell and tissue dynamical effects occurring from remodeling and/or drugs in heart failure. We demonstrate theoretically and experimentally in six different species the existence of a family of excitation wavelengths, we termed semasbestic, that give no change in signal for one dye, and thus can be used to record signals from another dye, guaranteeing zero cross-talk.

Intracellular Calcium Recording After Purinoceptor Activation Using a Video-Microscopy Equipment.

Calcium is one of the most important intracellular messengers, triggering a wide range of cellular responses. Changes in intracellular free calcium concentration can be measured using calcium sensitive fluorescent dyes, which are either EGTA- or BAPTA-based organic molecules that change their spectral properties in response to Ca2+ binding. One of the most common calcium indicators is the ratiometric dye Fura-2. The main advantage of using ratiometric dyes is that the ratio signal is independent of the illumination intensity, dye concentration, photobleaching, and focus changes among others, allowing for the concentration of intracellular calcium to be determined independently of these artifacts. In this protocol, we describe the use of Fura-2 to measure intracellular calcium elevations in single cultured cells after purinoceptor activation using a video-microscopy equipment. This method, usually known as calcium imaging, allows for real-time quantification of intracellular calcium dynamics and can be adapted to measure agonist mediated intracellular calcium responses due to the activation of different purinergic receptors in several cellular models using the appropriate growth conditions.

Simulated microgravity reduces intracellular-free calcium concentration by inhibiting calcium channels in primary mouse osteoblasts.

Calcium homeostasis in osteoblasts plays fundamental roles in the physiology and pathology of bone tissue. Various types of mechanical stimuli promote osteogenesis and increase bone formation elicit increases in intracellular-free calcium concentration in osteoblasts. However, whether microgravity, a condition of mechanical unloading, exerts an influence on intracellular-free calcium concentration in osteoblasts or what mechanisms may underlie such an effect are unclear. Herein, we show that simulated microgravity reduces intracellular-free calcium concentration in primary mouse osteoblasts. In addition, simulated microgravity substantially suppresses the activities of L-type voltage-sensitive calcium channels, which selectively allow calcium to cross the plasma membrane from the extracellular space. Moreover, the functional expression of ryanodine receptors and inositol 1,4,5-trisphosphate receptors, which mediate the release of calcium from intracellular storage, decreased under simulated microgravity conditions. These results suggest that simulated microgravity substantially reduces intracellular-free calcium concentration through inhibition of calcium channels in primary mouse osteoblasts. Our study may provide a novel mechanism for microgravity-induced detrimental effects in osteoblasts, offering a new avenue to further investigate bone loss induced by mechanical unloading.

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 Ca(2+) signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca(2+)]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 [Ca(2+)]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca(2+)]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 [Ca(2+)]i in human neutrophils was observed. In Ca(2+)-free buffer, NAC- and cysteine-induced [Ca(2+)]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca(2+)]i in human neutrophils occur through Ca(2+) influx. NAC- and cysteine-induced [Ca(2+)]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 [Ca(2+)]i in human neutrophils, arguing against the possibility that Na(+)-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca(2+)]i increasing activity. Our results show that NAC and cysteine induce [Ca(2+)]i increase through Ca(2+) influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

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 ([Ca(2+)]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 Ca(2+) infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 µM) and SKF-96365 (20 µM) signifi cantly inhibited K49-PLA2-induced [Ca(2+)]i increase. These results suggest that K49-PLA2 modulates [Ca(2+)]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.

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.

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 ([Ca(2+)]i) in these cells, although carbachol consistently increased [Ca(2+)]i. Exposure of cells to high temperature (>43℃) or acidic bath solution (pH5.4) did not increase [Ca(2+)]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.

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.

Synthesis and properties of Asante Calcium Red--a novel family of long excitation wavelength calcium indicators.

Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450-540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca2+ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca(2+)-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (< 490 nm) or multiphoton (∼780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca2+]i measurement in GFP expressing cells. The indicators bind Ca2+ with either high (Kd = 0.49 ± 0.07 µM; ACR-1) or low affinity (Kd = 6.65 ± 0.13 µM; ACR-1-LA). Chelating Zn2+ (Kd = 0.38 ± 0.02 nM) or Mg2+ (Kd∼5mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa = 6.31 ± 0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators.

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.

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.

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 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.

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

Ginsenoside Rg1 inhabits cardiomyocyte hypertrophy induced by prostaglandin F_(2?) / 中国药理学通报

Aim To study the effect of ginsenoside Rg1(Rg1)on cardio myocyte hypertrophy induced by prostaglandin F2?(PGF2?),and to probe primarily into its mechanism.Methods The cultured neonatal rat cardiomyocyte hypertrophic response and the antihypertrophic effects of Rg1 were observed by measuring the cell diameter,protein content and the expression of atrial natriuretic factor(ANF) mRNA,which was assayed by real-time PCR.For mechanism studies,the intracellular free calcium concentration(i) in cultured cardiomyocytes was measured by using Fura-2/AM as a fluorescent indicator,nitric oxide(NO) metabolite level in the culture medium was tested by using Nitric Oxide Synthase Detection Kit.Results PGF2? 0.1 ?mol?L-1 caused the increases in the cardiomyocyte cell diameter,protein content and the expression of ANF mRNA.It could increase the i in cultured cardiomyocytes.Rg1 15.6、31.2、62.4 ?mol?L-1 could concentration-dependently inhibit the cardiomyocyte hypertrophy induced by PGF2?,and the cell diameter of cardiomyocyte treated by PGF2? was decreased by 18.4%、32.7%、43.8%(P