In Situ Calibration of Cytoplasmic and Nucleoplasmic Calcium Concentration in Adult Rat and Mouse Cardiac Myocytes

This study examines the use of carboxy-seminaphthorhodafluor-1 (C-SNARF-1) as an indicator of cytosolic pH in isolated rat cardiac myocytes. The emission spectrum of C-SNARF-1 when excited at 530 nm contains two well-separated peaks at approximately 590 and 640 nm, corresponding to the acidic and basic forms of the indicator. This spectral feature allows the indicator to be used in the single excitation, dual emission ratio mode. When C-SNARF-1 is loaded into rat cardiac myocytes as the membrane permeant ester derivative, C-SNARF-1/AM, the indicator localizes within the cytosol with virtually no partitioning into the mitochondria. C-SNARF-1 does not load into isolated mitochondria in suspension. There was no evidence for the presence of non-deesterified C-SNARF-1 within the cells. C-SNARF-1 can be calibrated in situ using a technique that abolishes all transsarcolemmal pH gradients. A 0.7-unit shift in the apparent pK (pKapp = pK-log10) between the in vitro calibration and the in situ calibration is consistent with a change in beta (I640 to pH 9/I640 at pH 5) in the cytosolic environment (beta in situ/beta in vitro = 0.21) and not a change in the true pK of the indicator. The contribution of cellular autofluorescence to the total signal can be made negligible. There is no effect of C-SNARF-1 on the contractile properties of rat cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

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In situ calibration of fura-2 and BCECF fluorescence in adult rat ventricular myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1990.259.3.h973 ◽

1990 ◽

Vol 259 (3) ◽

pp. H973-H981 ◽

Cited By ~ 7

Author(s):

S. Borzak ◽

R. A. Kelly ◽

B. K. Kramer ◽

Y. Matoba ◽

J. D. Marsh ◽

...

Keyword(s):

Ph Value ◽

Calibration Procedure ◽

Fluorescence Signal ◽

Additional Advantage ◽

Fura 2 ◽

Adult Rat ◽

Ventricular Cells ◽

In Situ Calibration

Quantitation of Ca+ and H+ activities within cells using presently available fluorescent probes is optimal when the fluorescence signal is calibrated in situ after each experiment. Fura-2 and 2',7'-bis(2-carboxy-ethyl)-5,6-carboxyfluoroscein (BCECF) are difficult to calibrate in freshly dissociated adult cardiac myocytes because calibration procedures produce cellular hypercontracture. In situ calibration was accomplished in rat ventricular cells by saturating fura-2 with La3+, an agent known to produce myocardial relaxation. Since fura-2 has different spectral properties when complexed with La3+ than with Ca2+, scaling factors were defined in vitro and then verified by experiments in cultured neonatal myocytes. In adult rat myocytes using the La3+ method, intracellular Ca2+ concentration ([Ca2+]i) was 131 +/- 47 nM (n = 14) in quiescent cells; diastolic [Ca2+]i and systolic [Ca2+]i in myocytes stimulated at 1 Hz were 140 +/- 56 and 1,088 +/- 211 nM (n = 5), respectively. BCECF fluorescence was calibrated in situ by a method that prevented cellular hypercontracture and reported a pH value of 7.10 +/- 0.10 in cells stimulated at 1.5 Hz. An additional advantage of both methods is that the buffers employed prevented large changes in the redox state of intracellular pyridine nucleotides, thus preventing a change in cellular autofluorescence during the calibration procedure.

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Differentiated membrane specializations and myofibrillar breakdown and recovery in cultured adult cardiac myocytes treated with TPA and diacylglycerol

Journal of Cell Science ◽

10.1242/jcs.93.1.95 ◽

1989 ◽

Vol 93 (1) ◽

pp. 95-105

Author(s):

R.L. Moses ◽

W.C. Claycomb

Keyword(s):

Cardiac Myocytes ◽

Morphological Evidence ◽

Cardiac Muscle Cells ◽

Active Growth ◽

Transverse Tubular System ◽

Adult Rat ◽

Intercalated Discs ◽

Transmission Electron ◽

Adult Cardiac Myocytes

Cultured adult rat ventricular cardiac muscle cells were treated with either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or diacylglycerol (DAG) and observed by in situ transmission electron microscopy. Membrane specializations present in untreated cells (intercalated discs, transverse tubules, plasmalemmal couplings) were also present after TPA and DAG treatment. In the case of the transverse tubular system, there was morphological evidence for active growth. Our studies showed that myofilaments began to become disorganized after 12–24 h of TPA treatment and that after 2 days of exposure to TPA the breakdown of sarcomeres was essentially complete. Myocytes that were treated with TPA for 2 days and then allowed to recover in control medium for 5 days contained sarcomeres in various stages of reassembly. These data indicate that TPA-treated cardiac myocytes retain several membrane specializations, suggesting that there are separate controls for myofilament organization and the maintenance of these differentiated plasmalemmal regions. Furthermore, the ability of the myocytes to recover from TPA treatment may provide investigators with a useful model with which to study myofibrillogenesis.

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Fluid-phase endocytosis by in situ cardiac myocytes of rat atria

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.4.c986 ◽

1993 ◽

Vol 265 (4) ◽

pp. C986-C996 ◽

Cited By ~ 3

Author(s):

E. Page ◽

J. Upshaw-Earley ◽

G. E. Goings ◽

D. A. Hanck

Keyword(s):

Cardiac Myocytes ◽

Secretory Granules ◽

Fluid Phase ◽

Membrane Receptors ◽

Adrenergic Stimulation ◽

Experimental Conditions ◽

Adult Rat ◽

Fluid Phase Endocytosis

Fluid-phase endocytosis (FPE) associated with recycling of fused plasmalemma-secretory granules or membranes and/or membrane receptors by in situ cardiac myocytes was studied at 37 degrees C in vitro noncontracting adult rat atrial preparations. Measurements included 1) the volume (VS*) of the compartment consisting of presumptive endocytotic vesicles and the endosomes or lysosomes transiently in continuity with them (S*), which internalizes [14C]-sucrose but is inaccessible to simultaneously measured [methoxy-3H]inulin, 2) the kinetics of [14C]sucrose efflux from S*, and 3) morphometry to quantify interstitial space and non-heart muscle cells. Vs* (0.39 +/- 0.04 ml/g dry atrium for unstretched atria at 37 degrees C) was 1) variable over a 3.7-fold range under various experimental conditions, 2) significantly increased by neomycin or by lowering the temperature to 18 degrees C, and 3) significantly decreased by alpha 1-adrenergic stimulation. Analysis of sucrose efflux kinetics confirmed the presence of an intramyocytic sucrose-containing compartment. A smaller inulin-inaccessible sucrose space (S*) was also present in right ventricle. Thus, during FPE, vesicles and endosomes initially containing high (extracellular) Ca2+ and Cl- concentrations continually enter, circulate within, and undergo exocytosis from myocardial cells.

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Unsupervised optimization approach to in situ calibration of collaborative human-robot interaction tools

2020 IEEE International Conference on Multisensor Fusion and Integration for Intelligent Systems (MFI) ◽

10.1109/mfi49285.2020.9235229 ◽

2020 ◽

Author(s):

Bruno Maric ◽

Marsela Polic ◽

Tomislav Tabak ◽

Matko Orsag

Keyword(s):

Human Robot Interaction ◽

Optimization Approach ◽

Robot Interaction ◽

In Situ Calibration

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Performance of the SoLid reactor neutrino detector

EPJ Web of Conferences ◽

10.1051/epjconf/201921908003 ◽

2019 ◽

Vol 219 ◽

pp. 08003

Author(s):

Maja Verstraeten

Keyword(s):

Quality Control ◽

Energy Resolution ◽

Neutrino Detector ◽

Channel Response ◽

In Situ Calibration ◽

Reactor Neutrino ◽

Li Isotope ◽

Better Than

The SoLid Collaboration is currently operating a 1.6 ton neutrino detector near the Belgian BR2 reactor. Its main goal is the observation of the oscillation of electron antineutrinos to previously undetected flavour states. The highly segmented SoLid detector employs a compound scintillation technology based on PVT scintillator in combination with LiF-ZnS(Ag) screens containing the 6Li isotope. The experiment has demonstrated a channel-to-channel response that can be controlled to the level of a few percent, an energy resolution of better than 14% at 1 MeV, and a determination of the interaction vertex with a precision of 5 cm. This contribution highlights the major outcomes of the R&D program, the quality control during component manufacture and integration, the current performance and stability of the full-scale system, as well as the in-situ calibration of the detector with various radioactive sources.

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