Pacing rate, halothane, and BDM affect fura 2 reporting of [Ca2+]iin intact rat trabeculae

1997 ◽  
Vol 273 (6) ◽  
pp. C2046-C2056 ◽  
Author(s):  
Yandong Jiang ◽  
Fred J. Julian
Keyword(s):  
Fluorescence Emission ◽  
Fura 2 ◽  
Oxidation Reduction ◽  
Intracellular Ca ◽  
Butanedione Monoxime ◽  
Main Component ◽  
In Vivo Calibration ◽  
Substantial Rise

Experiments were done on intact trabeculae from rats. Fura 2 in the salt form was microinjected directly into the myoplasm. The experiments were conducted at 30°C, with 2 mM extracellular Ca2+ concentration and pacing at either 0.5 or 5 Hz. The aims were to establish a new method for in vivo calibration of fura 2 and to determine the effect of autofluorescence changes on intracellular Ca2+ concentration ([Ca2+]i) reported by fura 2. Autofluorescence was recorded under optimal conditions for fura 2 fluorescence (emission at 510 nm). By alteration of the oxidation-reduction state, it was shown that NADH is the main component of autofluorescence in heart. An increase in pacing frequency caused a decrease in autofluorescence. Both halothane and 2,3-butanedione monoxime (BDM) at 5-Hz pacing produced a substantial rise in autofluorescence, approaching the levels observed at 0.5-Hz pacing. The values for the dissociation constant (678 nM) and maximum fluorescence ratio of fura 2 for Ca2+ for the in vivo calibration are 3.4 times larger and 2.6 times smaller, respectively, than those found in vitro. Using the parameters obtained in vivo, we found that the diastolic and systolic [Ca2+]iof a twitch at 30°C were 0.2 and 2.4 μM, respectively. Proper correction of the autofluorescence change unmasks the [Ca2+]ielevation caused by 5-Hz pacing. It was concluded that autofluorescence is not constant and that interventions affecting autofluorescence need correction if fura 2 is used to report [Ca2+]i.

scholarly journals Temperature effects on morphological integrity and Ca2+ signaling in freshly isolated murine feed artery endothelial cell tubes

2011 ◽  
Vol 301 (3) ◽  
pp. H773-H783 ◽  
Author(s):  
Matthew J. Socha ◽  
Chady H. Hakim ◽  
William F. Jackson ◽  
Steven S. Segal
Keyword(s):  
Endothelial Cell ◽  
Enzymatic Digestion ◽  
Receptor Activation ◽  
Abdominal Muscle ◽  
Fura 2 ◽  
Intracellular Ca ◽  
Feed Arteries ◽  
In Vitro Stability

To study Ca2+ signaling in the endothelium of murine feed arteries, we determined the in vitro stability of endothelial cell (EC) tubes freshly isolated from abdominal muscle feed arteries of male and female C57BL/6 mice (5–9 mo, 25–35 g). We tested the hypothesis that intracellular Ca2+ concentration ([Ca2+]i) responses to muscarinic receptor activation would increase with temperature. Intact EC tubes (length: 1–2 mm, width: 65–80 μm) were isolated using gentle enzymatic digestion with trituration to remove smooth muscle cells. A freshly isolated EC tube was secured in a chamber and superfused at 24 (room temperature), 32, or 37°C. Using fura-2 dye, [Ca2+]i was monitored (ratio of fluorescence at 340- to 380-nm wavelength) at rest and in response to bolus doses of ACh (20 nmol to 200 μmol). The morphological integrity of EC tubes was preserved at 24 and 32°C. Based on the Ca2+ Kd values we determined for fura-2 (174 nM at 24°C and 146 nM at 32°C), resting [Ca2+]i remained stable for 180 min at both 24 and 32°C (27 ± 4 and 34 ± 2 nM, respectively), with peak responses to ACh (20 μmol) increasing from ∼220 nM at 24°C to ∼500 nM at 32°C ( P < 0.05). There was no difference in responses to ACh between EC tubes from male versus female mice. When EC tubes were maintained at 37°C (typical in vivo temperature), resting [Ca2+]i increased by ∼30% within 15 min, and gaps formed between individual ECs as they retracted and extruded dye, precluding further study. We conclude that EC tubes enable Ca2+ signaling to be evaluated in the freshly isolated endothelium of murine feed arteries. While Ca2+ responses are enhanced by approximately twofold at 32 versus 24°C, the instability of EC tubes at 37°C precludes their study at typical body temperature.

Enhanced Increases in Cytosolic Ca2+ in ADP-Stimulated Platelets from Patients with Delta-Storage Pool Deficiency – A Possible Indicator of Interactions between Granule-Bound ADP and the Membrane ADP Receptor

1997 ◽  
Vol 77 (02) ◽  
pp. 376-382 ◽  
Author(s):  
Bruce Lages ◽  
Harvey J Weiss
Keyword(s):  
Platelet Rich Plasma ◽  
Limited Range ◽  
Dense Granule ◽  
Receptor Desensitization ◽  
Fura 2 ◽  
Storage Pool ◽  
Low Levels ◽  
The Right

SummaryThe possible involvement of secreted platelet substances in agonist- induced [Ca2+]i increases was investigated by comparing these increases in aspirin-treated, fura-2-loaded normal platelets and platelets from patients with storage pool deficiencies (SPD). In the presence and absence of extracellular calcium, the [Ca2+]i response induced by 10 µM ADP, but not those induced by 0.1 unit/ml thrombin, 3.3 µM U46619, or 20 µM serotonin, was significantly greater in SPD platelets than in normal platelets, and was increased to the greatest extent in SPD patients with Hermansky-Pudlak syndrome (HPS), in whom the dense granule deficiencies are the most severe. Pre-incubation of SPD-HPS and normal platelets with 0.005-5 µM ADP produced a dose-dependent inhibition of the [Ca2+]i response induced by 10 µ M ADP, but did not alter the [Ca2+]i increases induced by thrombin or U46619. Within a limited range of ADP concentrations, the dose-inhibition curve of the [Ca2+]i response to 10 µM ADP was significantly shifted to the right in SPD-HPS platelets, indicating that pre-incubation with greater amounts of ADP were required to achieve the same extent of inhibition as in normal platelets. These results are consistent with a hypothesis that the smaller ADP-induced [Ca2+]i increases seen in normal platelets may result from prior interactions of dense granule ADP, released via leakage or low levels of activation, with membrane ADP receptors, causing receptor desensitization. Addition of apyrase to platelet-rich plasma prior to fura-2 loading increased the ADP-induced [Ca2+]i response in both normal and SPD-HPS platelets, suggesting that some release of ADP derived from both dense granule and non-granular sources occurs during in vitro fura-2 loading and platelet washing procedures. However, this [Ca2+]i response was also greater in SPD-HPS platelets when blood was collected with minimal manipulation directly into anticoagulant containing apyrase, raising the possibility that release of dense granule ADP resulting in receptor desensitization may also occur in vivo. Thus, in addition to enhancing platelet activation, dense granule ADP could also act to limit the ADP-mediated reactivity of platelets exposed in vivo to low levels of stimulation.

Paradoxical effect of salbutamol in a model of acute organophosphates intoxication in guinea pigs: role of substance P release

2007 ◽  
Vol 292 (4) ◽  
pp. L915-L923 ◽  
Author(s):  
Jaime Chávez ◽  
Patricia Segura ◽  
Mario H. Vargas ◽  
José Luis Arreola ◽  
Edgar Flores-Soto ◽  
...  
Keyword(s):  
Substance P ◽  
Guinea Pigs ◽  
Smooth Muscle Contraction ◽  
In Vivo Experiments ◽  
Intracellular Ca ◽  
Neurokinin 1 ◽  
Substance P Release

Organophosphates induce bronchoobstruction in guinea pigs, and salbutamol only transiently reverses this effect, suggesting that it triggers additional obstructive mechanisms. To further explore this phenomenon, in vivo (barometric plethysmography) and in vitro (organ baths, including ACh and substance P concentration measurement by HPLC and immunoassay, respectively; intracellular Ca2+ measurement in single myocytes) experiments were performed. In in vivo experiments, parathion caused a progressive bronchoobstruction until a plateau was reached. Administration of salbutamol during this plateau decreased bronchoobstruction up to 22% in the first 5 min, but thereafter airway obstruction rose again as to reach the same intensity as before salbutamol. Aminophylline caused a sustained decrement (71%) of the parathion-induced bronchoobstruction. In in vitro studies, paraoxon produced a sustained contraction of tracheal rings, which was fully blocked by atropine but not by TTX, ω-conotoxin (CTX), or epithelium removal. During the paraoxon-induced contraction, salbutamol caused a temporary relaxation of ∼50%, followed by a partial recontraction. This paradoxical recontraction was avoided by the M2- or neurokinin-1 (NK1)-receptor antagonists (methoctramine or AF-DX 116, and L-732138, respectively), accompanied by a long-lasting relaxation. Forskolin caused full relaxation of the paraoxon response. Substance P and, to a lesser extent, ACh released from tracheal rings during 60-min incubation with paraoxon or physostigmine, respectively, were significantly increased when salbutamol was administered in the second half of this period. In myocytes, paraoxon did not produce any change in the intracellular Ca2+ basal levels. Our results suggested that: 1) organophosphates caused smooth muscle contraction by accumulation of ACh released through a TTX- and CTX-resistant mechanism; 2) during such contraction, salbutamol relaxation is functionally antagonized by the stimulation of M2 receptors; and 3) after this transient salbutamol-induced relaxation, a paradoxical contraction ensues due to the subsequent release of substance P.

scholarly journals Sustainable, Rapid Synthesis of Bright-Luminescent CuInS2-ZnS Alloyed Nanocrystals: Multistage Nano-xenotoxicity Assessment and Intravital Fluorescence Bioimaging in Zebrafish-Embryos

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
S. Shashank Chetty ◽  
S. Praneetha ◽  
Sandeep Basu ◽  
Chetana Sachidanandan ◽  
A. Vadivel Murugan
Keyword(s):  
Large Scale ◽  
Near Infrared ◽  
Organic Dyes ◽  
Low Cost ◽  
Fluorescence Emission ◽  
Zebrafish Embryos ◽  
Fluorescence Bioimaging ◽  
Rapid Labeling

Abstract Near-infrared (NIR) luminescent CuInS2-ZnS alloyed nanocrystals (CIZS-NCs) for highly fluorescence bioimaging have received considerable interest in recent years. Owing, they became a desirable alternative to heavy-metal based-NCs and organic dyes with unique optical properties and low-toxicity for bioimaging and optoelectronic applications. In the present study, bright and robust CIZS-NCs have been synthesized within 5 min, as-high-as 230 °C without requiring any inert-gas atmosphere via microwave-solvothermal (MW-ST) method. Subsequently, the in vitro and in vivo nano-xenotoxicity and cellular uptake of the MUA-functionalized CIZS-NCs were investigated in L929, Vero, MCF7 cell lines and zebrafish-embryos. We observed minimal toxicity and acute teratogenic consequences upto 62.5 μg/ml of the CIZS-NCs in zebrafish-embryos. We also observed spontaneous uptake of the MUA-functionalized CIZS-NCs by 3 dpf older zebrafish-embryos that are evident through bright red fluorescence-emission at a low concentration of 7.8 μg/mL. Hence, we propose that the rapid, low-cost, large-scale “sustainable” MW-ST synthesis of CIZS-NCs, is an ideal bio-nanoprobe with good temporal and spatial resolution for rapid labeling, long-term in vivo tracking and intravital-fluorescence-bioimaging (IVBI).

Abstract 1079: Prevention of Fatal Arrhythmia in a Catecholaminergic Polymorphic Ventricular Tachycardia Mouse Model Carrying Calsequestrin-2 Mutation

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Hiroko Wakimoto ◽  
Ronny Alcalai ◽  
Lei Song ◽  
Michael Arad ◽  
Christine E Seidman ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Mouse Model ◽  
Peak Height ◽  
Mutant Mice ◽  
Catecholaminergic Polymorphic Ventricular Tachycardia ◽  
Polymorphic Ventricular Tachycardia ◽  
Study Drug Administration ◽  
Intracellular Ca

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmia syndrome caused by mutations in the ryanodine receptor (RyR2) or calsequestrin-2 (CASQ2) genes and characterized by exercise or emotional stress-induced sudden death. Beta-adrenergic blockers are only partially effective and other agents have not been widely tested. Recent studies have shown that CPVT is mediated by increased Ca 2+ leak through the RyR2 channel. Our aim was to determine whether agents that inhibit intracellular Ca 2+ leak can effectively prevent CPVT. Methods: The efficacy of intraperitoneal (IP) propranolol (1mcg/g), Mg 2+ (0.002mEq/g), verapamil (8 mcg/g) and diltiazem (8 mcg/g) were tested both in vivo and in vitro using CASQ2 mutant mouse CPVT model. In vivo studies included ambulatory ECG recordings at rest and following epinephrine stress (0.4 mcg/g IP) at baseline and after study drug administration. Experiments for each drug were performed on separate days to avoid confounding effects. In vitro studies included intracellular Ca 2+ transient analysis on isolated cardiomyocytes from mutant mice with and without epinephrine (5.5 μM). Results: All 4 drugs restored sinus rhythm and reduced the frequency of VT episodes induced by epinephrine in CASQ2 mutant mice. Only verapamil completely prevented epinephrine-induced VT in 87% of the mice (p<0.01). Cardiomyocyte studies in basal conditions revealed that Mg 2+ and verapamil inhibited sarcomere contraction and normalized the prolonged Ca 2+ reuptake period in CASQ2 mutants, but did not decrease baseline Ca 2+ peak height. Epinephrine-stressed mutant myocytes had increased diastolic Ca 2+ levels, lower Ca 2+ peak height and spontaneous SR Ca 2+ release events that were partially prevented by verapamil and Mg 2+ . Verapamil was more effective than Mg 2+ in reducing the frequency of spontaneous Ca 2+ releases induced by epinephrine. Conclusions: All 4 agents can inhibit ventricular arrhythmia in CPVT mouse model; however verapamil appears most effective in preventing arrhythmia in vivo and in modifying intracellular abnormal calcium handling. Calcium antagonists might have therapeutic value in CPVT and other RyR2-mediated arrhythmias and should be considered for human clinical studies.

A multipurpose instrument for quantitative intravital microscopy

1992 ◽  
Vol 73 (1) ◽  
pp. 296-306 ◽  
Author(s):  
A. Toth ◽  
M. E. Tischler ◽  
M. Pal ◽  
A. Koller ◽  
P. C. Johnson
Keyword(s):  
Parenchymal Cell ◽  
Fluorescence Emission ◽  
Repeated Measurements ◽  
Open Architecture ◽  
In Vitro Tests ◽  
Fluorescence Measurements ◽  
Tissue Area ◽  
Microcirculatory Function

An in vivo microscope system has been developed that can measure fluorescence emission and/or light absorption at up to five wavelengths in a tissue area of 18–30 microns diam while imaging adjacent microcirculatory vessels with a video system. The system also incorporates a computer-controlled stage and data acquisition system for rapid and repeated measurements from a number of tissue sites. The tissue area monitored for fluorescence or absorption can be defined further by a confocal arrangement of the microscope optics. Tests of the system for NADH fluorescence measurements show good agreement between the fluorescence at 450 nm and NADH concentration in vitro and in skeletal muscle. The instrument can also be used simultaneously for spectrophotometric determination of O2 saturation and hematocrit in microcirculatory vessels. In vitro tests indicate suitable accuracy for such measurements. The open architecture and modular arrangement of the instrument facilitates its use for a variety of simultaneous measurements of parenchymal cell and microcirculatory function.

scholarly journals ADME/Tox Properties and Biochemical Interactions of Silybin Congeners: In silico Study

2017 ◽  
Vol 12 (2) ◽  
pp. 1934578X1701200 ◽  
Author(s):  
Antonia Diukendjieva ◽  
Merilin Al Sharif ◽  
Petko Alov ◽  
Tania Pencheva ◽  
Ivanka Tsakovska ◽  
...  
Keyword(s):  
In Silico ◽  
Estrogen Receptor Alpha ◽  
Estrogenic Activity ◽  
Qsar Model ◽  
Quantitative Structure Activity Relationship ◽  
Toxic Effects ◽  
Active Constituent ◽  
Main Component

Silymarin, the active constituent of Silybum marianum (milk thistle), and its main component, silybin, are products with well-known hepatoprotective, cytoprotective, antioxidant, and chemopreventative properties. Despite substantial in vitro and in vivo investigations of these flavonolignans, their mechanisms of action and potential toxic effects are not fully defined. In this study we explored important ADME/Tox properties and biochemical interactions of selected flavonolignans using in silico methods. A quantitative structure–activity relationship (QSAR) model based on data from a parallel artificial membrane permeability assay (PAMPA) was used to estimate bioavailability after oral administration. Toxic effects and metabolic transformations were predicted using the knowledge-based expert systems Derek Nexus and Meteor Nexus (Lhasa Ltd). Potential estrogenic activity of the studied silybin congeners was outlined. To address further the stereospecificity of this effect the stereoisomeric forms of silybin were docked into the ligand-binding domain of the human estrogen receptor alpha (ERα) (MOE software, CCG). According to our results both stereoisomers can be accommodated into the ERα active site, but different poses and interactions were observed for silybin A and silybin B.

Ratiometric measurement of endothelial depolarization in arterioles with a potential-sensitive dye

1996 ◽  
Vol 270 (6) ◽  
pp. H2216-H2227 ◽  
Author(s):  
J. M. Beach ◽  
E. D. McGahren ◽  
J. Xia ◽  
B. R. Duling
Keyword(s):  
Membrane Potential ◽  
Fluorescence Emission ◽  
Cheek Pouch ◽  
Voltage Sensitivity ◽  
Hamster Cheek Pouch ◽  
Endothelial Cell Layer ◽  
Ratiometric Measurement ◽  
Length Constant

A fluorescence ratio technique based on the voltage-sensitive dye 1-(3-sulfonatopropyl)-8-[beta-[2-di-n-butylamino)-6-naphythyl++ +]vinyl] pyridinium betaine (di-8-ANEPPS)has been developed for recording membrane potential changes during vascular responses of arterioles. Perfusion of hamster cheek pouch arterioles with the dye labeled the endothelial cell layer. voltage responses from the endothelium of intact arterioles were determined by analysis of voltage-induced shifts in fluorescence emission wavelengths from dye spectra imaged from the vessel wall. Membrane depolarization caused the dye spectrum to shift toward blue wavelengths, with maximal fluorescence changes near 560 and 620 nm. In isolated nonperfused arterioles, comparison of continuous dual-wavelength recordings with simultaneous microelectrode recordings showed that the ratio of fluorescence intensities (fluorescence at 620 nm to fluorescence at 560 nm) accurately followed changes in membrane potential (6–21 mV) during vasoconstriction. The dye response was linear with respect to potential changes from -56 to -6 mV, with a voltage sensitivity of 9.7% change in the ratio per 100 mV. Membrane potential responses from in vitro and in vivo arterioles after potassium stimulation consisted of rapid ( < 0.5 -s) depolarization followed by slow repolarization over several seconds. Potassium-induced depolarizations were conducted along arterioles, and the values of the electrical length constant for conducted depolarization determined by optical and microelectrode methods were in agreement. We conclude that ratio analysis of di-8-ANEPPS fluorescence emission can be used to accurately record membrane potential changes on the time scale of seconds during vasomotor activity from arterioles.

scholarly journals Echinacea angustifolia DC. Lipophilic Extract Patch for Skin Application: Preparation, In Vitro and In Vivo Studies

Pharmaceutics ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1096
Author(s):  
Dritan Hasa ◽  
Simon Žakelj ◽  
Iztok Grabnar ◽  
Francesco Cilurzo ◽  
Stefano Dall’Acqua ◽  
...  
Keyword(s):  
Healthy Volunteers ◽  
Skin Permeation ◽  
In Vivo Studies ◽  
Pharmacokinetic Analysis ◽  
Echinacea Angustifolia ◽  
Transdermal Administration ◽  
Lipophilic Extract ◽  
Main Component

Dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamide (tetraene) is the main component of Echinacea angustifolia DC. lipophilic extract, the bioavailability and immunomodulatory effect after oral administration in soft gel capsules in healthy volunteers of which we have already demonstrated. In the present work, we assessed the transdermal administration as an alternative route of administration of such an alkamide. The first step, therefore, encompassed the preparation of a drug-in-adhesive patch with an area of 868 mm2 and containing a dose of 0.64 mg of tetraene. In vitro skin permeation studies in Franz-type diffusion chambers resulted in a tetraene flux of (103 ± 10) ng × cm−2 × h−1 with a very good linearity (r = 0.99). The relatively low lag time of just 13 min indicates low binding and the accumulation of tetraene in the skin. Finally, the patch was administered to six healthy volunteers, and the pharmacokinetic analysis was performed by nonlinear mixed effects modelling with soft gel oral capsules serving as the reference formulation. The in vivo results correlated well with the in vitro permeation and indicated an initial burst tetraene absorption from the patch that was in parallel with the zero-order kinetics of absorption. The rate of the latter process was in good agreement with the one estimated in vitro. The tetraene absorption rate was therefore slow and prolonged with time, resulting in a bioavailability of 39% relative to the soft gel capsules and a very flat plasma concentration profile.

scholarly journals Mechanisms for Intracellular Calcium Regulation in Heart

1973 ◽  
Vol 62 (6) ◽  
pp. 756-772 ◽  
Author(s):  
Antonio Scarpa ◽  
Pierpaolo Graziotti
Keyword(s):  
Cardiac Cells ◽  
Heart Mitochondria ◽  
Frog Heart ◽  
Cardiac Mitochondria ◽  
Physiological Regulation ◽  
Ca Transport ◽  
Ca Uptake ◽  
Intracellular Ca

Initial velocities of energy-dependent Ca++ uptake were measured by stopped-flow and dual-wavelength techniques in mitochondria isolated from hearts of rats, guinea pigs, squirrels, pigeons, and frogs. The rate of Ca++ uptake by rat heart mitochondria was 0.05 nmol/mg/s at 5 µM Ca++ and increased sigmoidally to 8 nmol/mg/s at 200 µM Ca++. A Hill plot of the data yields a straight line with slope n of 2, indicating a cooperativity for Ca++ transport in cardiac mitochondria. Comparable rates of Ca++ uptake and sigmoidal plots were obtained with mitochondria from other mammalian hearts. On the other hand, the rates of Ca++ uptake by frog heart mitochondria were higher at any Ca++ concentrations. The half-maximal rate of Ca++ transport was observed at 30, 60, 72, 87, 92 µM Ca++ for cardiac mitochondria from frog, squirrel, pigeon, guinea pig, and rat, respectively. The sigmoidicity and the high apparent Km render mitochondrial Ca++ uptake slow below 10 µM. At these concentrations the rate of Ca++ uptake by cardiac mitochondria in vitro and the amount of mitochondria present in the heart are not consistent with the amount of Ca++ to be sequestered in vivo during heart relaxation. Therefore, it appears that, at least in mammalian hearts, the energy-linked transport of Ca++ by mitochondria is inadequate for regulating the beat-to-beat Ca++ cycle. The results obtained and the proposed cooperativity for mitochondrial Ca++ uptake are discussed in terms of physiological regulation of intracellular Ca++ homeostasis in cardiac cells.

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