Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells

Subedi, Krishna Prasad; Paudel, Omkar; Sham, James S.K.

doi:10.1152/ajpcell.00341.2013

Cited by 10 publications

(7 citation statements)

References 66 publications

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“…This "in vivtro" method [Narang et al, 1986] falls somewhere between truly in vitro methods where all the constituents of a system are known, and in vivo methods where the system being assayed is living and consists solely of endogenous components. Similar to Xenopus egg extract systems, it is clear that some cellular activities will be disrupted by establishment of the assay system while other activities, such as nuclear import [Turgay et al, 2010], calcium signaling [Subedi et al, 2014], cardiomyocyte contractility [Altschuld et al, 1985], and platelet function [Lineberger et al, 1989], remain intact. Although PARF may be applicable to the study of a specific subset of cellular processes, the technical capabilities required for PARF are less than those required for other quantitative imaging techniques (such as photoactivation or FRAP), nor does PARF require the technical expertize in protein expression and purification necessary for reconstitution approaches.…”

Section: Parf Is a Flexible And Useful Technique Capable Of Addressinmentioning

confidence: 99%

“…Permeabilization has been used to allow manipulation of cytosolic ion concentrations [Altschuld et al, 1985;Smolen et al, 1986], to allow introduction of large macromolecules [Lorenz et al, 2008], and to allow removal of macromolecules [Lineberger et al, 1989]. In many of these experiments, the cell structures that remained following digitonin permeabilization continued to behave similarly to intact cells, allowing researchers to characterize subcellular processes using approaches not available in intact cells or in in vitro reconstitution systems [Marino et al, 2014;Schulz, 1990;Subedi et al, 2014].…”

Section: Introductionmentioning

confidence: 99%

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Permeabilization activated reduction in fluorescence: A novel method to measure kinetics of protein interactions with intracellular structures

et al. 2016

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Understanding kinetic information is fundamental in understanding biological function. Advanced imaging technologies have fostered the development of kinetic analyses in cells. We have developed Permeabilization Activated Reduction in Fluorescence (PARF) analysis for determination of apparent t1/2 and immobile fraction, describing the dissociation of a protein of interest from intracellular structures. To create conditions where dissociation events are observable, cells expressing a fluorescently-tagged protein are permeabilized with digitonin, diluting the unbound protein into the extracellular media. As the media volume is much larger than the cytosolic volume, the concentration of the unbound pool decreases drastically, shifting the system out of equilibrium--favoring dissociation events. Loss of bound protein is observed as loss of fluorescence from intracellular structures and can be fit to an exponential decay. We compared PARF dissociation kinetics with previously published equilibrium kinetics as determined by FRAP. PARF dissociation rates agreed with the equilibrium-based FRAP analysis predictions of the magnitude of those rates. When used to investigate binding kinetics of a panel of cytoskeletal proteins, PARF analysis revealed that filament stabilization resulted in slower fluorescence loss. Additionally, commonly used “general” F-actin labels display differences in kinetic properties, suggesting that not all fluorescently-tagged actin labels interact with the actin network in the same way. We also observed differential dissociation kinetics for GFP-VASP depending on which cellular structure was being labeled. These results demonstrate that PARF analysis of non-equilibrium systems reveals kinetic information without the infrastructure investment required for other quantitative approaches such as FRAP, photoactivation, or in vitro reconstitution assays.

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Section: Parf Is a Flexible And Useful Technique Capable Of Addressinmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Permeabilization activated reduction in fluorescence: A novel method to measure kinetics of protein interactions with intracellular structures

et al. 2016

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“…Rather, calcium signals are highly dynamic and reliant on a combination of deterministic and stochastic processes. Our laboratory, and others', have previously reported that pulmonary arterial myocytes exhibit whole cell Ca 2ϩ events that are spontaneous or can be activated by various forms of stimulation (34,38,43,46). Although Ca L activation and inhibition affect pulmonary arterial myocyte contractions by modifying intracellular Ca 2ϩ events, the coupling between cell activation, Ca 2ϩ responses, and the resulting myocyte contractions is not completely understood (46).…”

Section: Introductionmentioning

confidence: 98%

Long-term high-altitude hypoxia influences pulmonary arterial L-type calcium channel-mediated Ca²⁺ signals and contraction in fetal and adult sheep

Shen

Romero

Brunelle

et al. 2018

American Journal of Physiology-Regulatory, Integrative and Comp

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Long-term hypoxia (LTH) has a profound effect on pulmonary arterial vasoconstriction in the fetus and adult. Dysregulation in Ca signaling is important during the development of LTH-induced pulmonary hypertension. In the present study, we tested the hypothesis that L-type Ca channels (Ca), which are voltage dependent and found in smooth, skeletal, and cardiac muscle, are important in the adaptation of pulmonary arterial contractions in postnatal maturation and in response to LTH. Pulmonary arteries were isolated from fetal or adult sheep maintained at low or high altitude (3,801 m) for >100 days. The effects were measured using an L-type Ca channel opener FPL 64176 (FPL) in the presence or absence of an inhibitor, Nifedipine (NIF) on arterial contractions, intracellular Ca oscillations, and ryanodine receptor-driven Ca sparks. FPL induced pulmonary arterial contractions in all groups were sensitive to NIF. However, when compared with 125 mM K, FPL contractions were greater in fetuses than in adults. FPL reduced Ca oscillations in myocytes of adult but not fetal arteries, independently of altitude. The FPL effects on Ca oscillations were reversed by NIF in myocytes of hypoxic but not normoxic adults. FPL failed to enhance Ca spark frequency and had little impact on spatiotemporal firing characteristics. These data suggest that Ca-dependent contractions are largely uncoupled from intracellular Ca oscillations and the development of Ca sparks. This raises questions regarding the coupling of pulmonary arterial contractility to membrane depolarization, attendant Ca facilitation, and the related associations with the activation of Ca oscillations and Ca sparks.

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“…Their divalent character makes them strong binders; hence, their physiological concentration must be strictly controlled. The concentration of Ca 2+ in the extracellular space is about 2 mM, while its intracellular levels are much lower and range amongst cell organelles from 100 nM in the cytosol to 600 μM in the endoplasmic reticulum12. The maintenance of low intracellular calcium concentrations is achieved, among other mechanisms, by calcium buffers which include both cytoplasmic and membrane-anchored calcium-binding proteins134.…”

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confidence: 99%

The complex nature of calcium cation interactions with phospholipid bilayers

Melcrová

Pokorná

Pullanchery

et al. 2016

Sci Rep

231

288

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Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

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Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells

Abstract: Subedi KP, Paudel O, Sham JS. Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells.

Cited by 10 publications

References 66 publications

Permeabilization activated reduction in fluorescence: A novel method to measure kinetics of protein interactions with intracellular structures

Permeabilization activated reduction in fluorescence: A novel method to measure kinetics of protein interactions with intracellular structures

Long-term high-altitude hypoxia influences pulmonary arterial L-type calcium channel-mediated Ca²⁺ signals and contraction in fetal and adult sheep

The complex nature of calcium cation interactions with phospholipid bilayers

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Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells

Abstract: Subedi KP, Paudel O, Sham JS. Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells.

Cited by 10 publications

References 66 publications

Permeabilization activated reduction in fluorescence: A novel method to measure kinetics of protein interactions with intracellular structures

Permeabilization activated reduction in fluorescence: A novel method to measure kinetics of protein interactions with intracellular structures

Long-term high-altitude hypoxia influences pulmonary arterial L-type calcium channel-mediated Ca2+ signals and contraction in fetal and adult sheep

The complex nature of calcium cation interactions with phospholipid bilayers

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Product

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Long-term high-altitude hypoxia influences pulmonary arterial L-type calcium channel-mediated Ca²⁺ signals and contraction in fetal and adult sheep