Ca2+ signals obtained with multiple indicators in mammalian vascular muscle cells

1987 ◽  
Vol 253 (6) ◽  
pp. H1456-H1461
Author(s):  
T. T. DeFeo ◽  
G. M. Briggs ◽  
K. G. Morgan
Keyword(s):  
Portal Vein ◽  
Calcium Concentration ◽  
Fluorescent Dyes ◽  
Contractile Function ◽  
Single Cells ◽  
Fluorescent Intensity ◽  
Fura 2 ◽  
Multiple Indicators ◽  
Calcium Storage ◽  
Vascular Muscle Cells

Enzymatically isolated single cells from ferret portal vein were loaded with the fluorescent dyes fura-2 and chlortetracycline. Ferret portal vein intact strips were loaded with the luminescent indicator aequorin. At short loading times, fura-2 loading resulted in relatively homogeneous images of labeled cells. At longer loading times, extremely heterogeneous images were obtained that were similar to those produced by chlortetracycline, an indicator recognized to enter calcium-storage organelles. A significant effect of fura-2 on contractile function was detected at the long but not at the short loading time. Caffeine, which is known to deplete calcium from sarcoplasmic reticulum, decreased the fura-2 fluorescent intensity when cells were incubated for a long loading time but caused no statistically significant change at the short loading time. Caffeine caused no drop in the aequorin signal but did cause a drop in the chlortetracycline fluorescence. These results are consistent with the idea that aequorin reports cytoplasmic intracellular calcium concentration ([Ca2+]i), chlortetracycline reports stored calcium, and fura-2 reports a mixed signal from the cytoplasm and calcium-storage organelles depending on incubation time.

scholarly journals Computational modeling of Takotsubo cardiomyopathy: effect of spatially varying β-adrenergic stimulation in the rat left ventricle

2014 ◽  
Vol 307 (10) ◽  
pp. H1487-H1496 ◽  
Author(s):  
Sander Land ◽  
Steven A. Niederer ◽  
William E. Louch ◽  
Åsmund T. Røe ◽  
Jan Magnus Aronsen ◽  
...  
Keyword(s):  
Left Ventricle ◽  
Takotsubo Cardiomyopathy ◽  
Troponin I ◽  
Contractile Function ◽  
Single Cells ◽  
Apical Ballooning ◽  
Calcium Sensitivity ◽  
Takotsubo Syndrome ◽  
Adrenergic Stimulation ◽  
Inotropic Effects

In Takotsubo cardiomyopathy, the left ventricle shows apical ballooning combined with basal hypercontractility. Both clinical observations in humans and recent experimental work on isolated rat ventricular myocytes suggest the dominant mechanisms of this syndrome are related to acute catecholamine overload. However, relating observed differences in single cells to the capacity of such alterations to result in the extreme changes in ventricular shape seen in Takotsubo syndrome is difficult. By using a computational model of the rat left ventricle, we investigate which mechanisms can give rise to the typical shape of the ventricle observed in this syndrome. Three potential dominant mechanisms related to effects of β-adrenergic stimulation were considered: apical-basal variation of calcium transients due to differences in L-type and sarco(endo)plasmic reticulum Ca2+-ATPase activation, apical-basal variation of calcium sensitivity due to differences in troponin I phosphorylation, and apical-basal variation in maximal active tension due to, e.g., the negative inotropic effects of p38 MAPK. Furthermore, we investigated the interaction of these spatial variations in the presence of a failing Frank-Starling mechanism. We conclude that a large portion of the apex needs to be affected by severe changes in calcium regulation or contractile function to result in apical ballooning, and smooth linear variation from apex to base is unlikely to result in the typical ventricular shape observed in this syndrome. A failing Frank-Starling mechanism significantly increases apical ballooning at end systole and may be an important additional factor underpinning Takotsubo syndrome.

Axotomy induces a transient and localized elevation of the free intracellular calcium concentration to the millimolar range

1995 ◽  
Vol 74 (6) ◽  
pp. 2625-2637 ◽  
Author(s):  
N. E. Ziv ◽  
M. E. Spira
Keyword(s):  
Intracellular Calcium ◽  
Calcium Concentration ◽  
Intracellular Calcium Concentration ◽  
Concentration Gradients ◽  
Fura 2 ◽  
Direct Demonstration ◽  
Free Intracellular Calcium ◽  
Axonal Transection ◽  
Transient Elevation

1. Axonal transection triggers a cascade of pathological processes that frequently lead to the degeneration of the injured neuron. It is generally believed that the degenerative process is triggered by an overwhelming influx of calcium through the cut end of the axon. 2. Theoretical considerations and indirect observations suggest that axotomy is followed by an increase in the free intracellular calcium concentration ([Ca2+]i) to the millimolar level. In contrast, only relatively modest and transient elevation in [Ca2+]i to the micromolar level was revealed by recent fura-2 studies. 3. In the current study we used the low-affinity Ca2+ indicator mag-fura-2 to reexamine the spatiotemporal distribution pattern of Ca2+ after axotomy and to map the free intracellular Mg2+ concentration gradients. 4. We report that axotomy elevates [Ca2+]i well beyond the "physiological" range of calcium concentrations, to levels > 1 mM near the tip of the cut axon and to hundreds of micromolars along the axon further away from the cut end. Nevertheless, [Ca2+]i recovers to the control levels within 2-3 min after the resealing of the cut end. 5. A comparison of the behavior of fura-2 and mag-fura-2 in the cytosol of the axotomized neurons reveals that the determination of [Ca2+]i by fura-2 largely underestimates the actual intracellular Ca2+ concentrations. 6. Experiments in which one branch of a bifurcated axon was transected revealed that the elevation in [Ca2+]i is confined to the transected axonal branch and does not spread beyond the bifurcation point. 7. After axotomy, the intracellular Mg2+ concentration equilibrates rapidly with the external concentration and then recovers at a rate somewhat slower than that of [Ca2+]i. 8. To the best of our knowledge, this study is the first direct demonstration that axotomy elevates [Ca2+]i to the millimolar range and that neurons are able to recover from these extreme calcium concentrations.

Changes in cytosolic cGMP and calcium in airway smooth muscle relaxed by 3-morpholinosydnonimine

1994 ◽  
Vol 266 (1) ◽  
pp. L9-L16 ◽  
Author(s):  
K. A. Jones ◽  
R. R. Lorenz ◽  
D. O. Warner ◽  
Z. S. Katusic ◽  
G. C. Sieck
Keyword(s):  
Methylene Blue ◽  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Tracheal Smooth Muscle ◽  
Relative Importance ◽  
Cytosolic Calcium Concentration ◽  
Fura 2 ◽  
Cyclic Monophosphate

Nitrovasodilators relax airway smooth muscle by both guanosine 3',5'-cyclic monophosphate (cGMP)-dependent and cGMP-independent mechanisms and by mechanisms that reduce cytosolic calcium concentration ([Ca2+]i). This study was conducted to determine the relative importance of these mechanisms in relaxation of canine tracheal smooth muscle (CTSM) induced by 3-morpholinosydnonimine (SIN-1). We measured 1) the effect of SIN-1 on force, [cGMP]i, and [Ca2+]i, and 2) the ability of methylene blue (MB) to antagonize SIN-1-induced relaxation and cGMP accumulation. The ratio of fura 2 emission fluorescence intensities due to excitation at 340- and 380-nm wavelengths (F340/F380) was used as an index of [Ca2+]i. In strips contracted with 0.3 microM acetylcholine (ACh, n = 8) or 24 mM KCl (n = 8), SIN-1 (1-100 microM) caused a concentration-dependent decrease in force which was correlated with a concentration-dependent increase in [cGMP]i. MB (10 microM) proportionally attenuated both relaxation and cGMP accumulation. In fura 2-loaded strips contracted with 0.3 microM ACh (n = 7) or 30 mM KCl (n = 7), reductions in force induced by SIN-1 (1-100 microM) were accompanied by decreases in F340/F380. These findings suggest that in CTSM contracted with ACh or KCl, SIN-1 causes relaxation which appears to be mediated by cGMP-dependent mechanisms that reduce [Ca2+]i.

Shortening and [Ca2+] dynamics of left ventricular myocytes isolated from exercise-trained rats

1998 ◽  
Vol 85 (6) ◽  
pp. 2159-2168 ◽  
Author(s):  
Bradley M. Palmer ◽  
Anne M. Thayer ◽  
Steven M. Snyder ◽  
Russell L. Moore
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Contractile Function ◽  
Left Ventricular ◽  
Trained Group ◽  
Effective Coupling ◽  
Temporal Characteristics ◽  
Fura 2 ◽  
Intracellular Ca ◽  
Maximal Extent

The effects of run endurance training and fura 2 loading on the contractile function and Ca2+ regulation of rat left ventricular myocytes were examined. In myocytes not loaded with fura 2, the maximal extent of myocyte shortening was reduced with training under our pacing conditions [0.5 Hz at 2.0 and 0.75 mM external Ca2+ concentration ([Ca2+]o)], although training had no effect on the temporal characteristics. The “light” loading of myocytes with fura 2 markedly suppressed (∼50%) maximal shortening in the sedentary and trained groups, although the temporal characteristics of myocyte shortening were significantly prolonged in the trained group. No discernible differences in the dynamic characteristics of the intracellular Ca2+ concentration ([Ca2+]) transient were detected at 2.0 mM [Ca2+]o, although peak [Ca2+] and rate of [Ca2+] rise during caffeine contracture were greater in the trained state at 0.75 mM [Ca2+]o. We conclude that training induced a diminished myocyte contractile function under the conditions studied here and a more effective coupling of inward Ca2+ current to sarcoplasmic reticulum Ca2+ release at low [Ca2+]o, and that fura 2 and its loading vehicle DMSO significantly alter the intrinsic characteristics of myocyte contractile function and Ca2+ regulation.

Cytoplasmic free calcium in single cells of centric diatoms. The use of fura-2

PROTOPLASMA ◽  
1987 ◽  
Vol 140 (2-3) ◽  
pp. 118-122 ◽  
Author(s):  
C. Brownlee ◽  
J. W. Wood ◽  
D. Briton
Keyword(s):  
Single Cells ◽  
Free Calcium ◽  
Fura 2 ◽  
Centric Diatoms ◽  
Cytoplasmic Free Calcium

Multicenter performance evaluation of the MultiDrugQuant assay kit.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e21140-e21140
Author(s):  
Peter Krajcsi ◽  
Katalin Tauber Jakab ◽  
Sandor Barath ◽  
Edit Gyimesi ◽  
Zsuzsanna Hevessy ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Mononuclear Cells ◽  
Fluorescent Dyes ◽  
Clinical Laboratory ◽  
Diagnostic Methods ◽  
Cytotoxic Agents ◽  
Efflux Transporters ◽  
Target Cells ◽  
Functional Assay ◽  
Fluorescent Intensity

e21140 Background: Multidrug resistance is the most frequent type of resistance to anticancer chemotherapy, which usually results from the overexpression of efflux transporters, such as the MDR1, MRP1 and BCRP. Unfortunatelly, neither the genetic polymorphisms nor the mRNA/protein expression levels correlate closely with the functional activity. On the other hand, although the functional methods separately gave promising results, standardization and reproducibility of these tests failed to conform with values required from routine diagnostic methods. MultiDrugQuant (MDQ) kit was developed as an improved functional assay system, which can measure the multidrug resistance activity of the three, clinically most relevant efflux transporters using flow cytometry in living tumor cells. The present study aimed to carry out the laboratory validation and to evaluate the performance of the MDQ-kit. Methods: Validation of the kit was carried out according to the standards of the Clinical Laboratory Standards Institute in three university centres. Mononuclear cells were separated using Ficoll gradient and tested at 2-5×106/ml within 6 hours after specimen collection. Activities of the multidrug transporters were calculated from the difference between the mean fluorescent intensity of cells w/o the specific inhibitors, respectively. Inaccuracy and comparative measurements were carried out using cell lines with low and high activity of the transporters. Results on different flow cytometers were compared using CD45 CD19 or CD3 monoclonal antibodies for gating the population of interest. Results: The assay proved to be specific and robust at various concentrations of the fluorescent dyes (10-100 % of the original) or inhibitors (50-150 % of the original). Both intraassay and interassay reproducibilities were <5 %. Multidrug resistance activity values determined on different flow cytometers were comparable and eligible. Conclusions: The MDQ assay provides quantitative results on the activity of the MDR1, MRP1 and BCRP in the target cells, which might be used to predict the resistance of these cells to particular cytotoxic agents. Recently, the MDQ-kit has been registered for in vitro diagnostic use in the EU.

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