<p class="jove_content"&gt; Este trabajo fue apoyado por el NIH subvención P20RR018766 y una subvención de la ABMRF / Fundación para la Investigación del Alcohol.</p

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Una nueva combinación de métodos se emplean para el estudio intrínseco de bombeo de los vasos linfáticos. La capacidad de medir simultáneamente los cambios en la [Ca 2 +] i y el diámetro de los vasos linfáticos de bombeo permitirá que para el estudio de las contribuciones relativas de Ca 2 +-dependiente y Ca 2 +-sensibilizar a las vías de señalización en el mecanismo general que rigen el ciclo de contracción linfático. El ciclo de cont...

<table border="1"><tbody><tr><td> 1. Ringer 5x de archivo </td></tr><tr><td Químico </td><td> Empresa </td><td> Número de catálogo </td><td> Cantidad </td></tr><tr><td> Cloruro de sodio </td><td> EMD </td><td> SX0420-3 </td><td> 35 g </td></tr><tr><td> Cloruro de Potasio </td><td> JT Baker </td><td> 3040 </td><td> 1,75 g </td></tr><tr><td> Cloruro de calcio </td><td> Sigma </td><td> C-3881 </td><td> 1,47 g </td></tr><tr><td> Sulfato de magnesio </td><td> Sigma </td><td> M-9397 </td><td> 1,44 g </td></tr><tr><td> El agua filtrada estéril </td><td> N / A </td><td> N / A </td><td> Llevar a 1 L </td></tr><tr><td> Filtro estéril en botellas autoclave y se almacena a 4 º C </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 2. MOPS buffer </td><td></td><td></td><td></td></tr><tr><td> Químico </td><td> Empresa </td&gt; <td> Número de catálogo </td><td> Cantidad </td></tr><tr><td> MOPS </td><td> Sigma </td><td> M3183 </td><td> 125,6 g </td></tr><tr><td> El agua filtrada estéril </td><td> N / A </td><td> N / A </td><td> Llevar a 1 L </td></tr><tr><td> Filtro estéril en botellas autoclave y se almacena a 4 º C </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 3. Solución de albúmina salina fisiológica (APSS) </td></tr><tr><td> Químico </td><td> Empresa </td><td> Número de catálogo </td><td> Cantidad </td></tr><tr><td> Timbre de valores (5x) </td><td> N / A </td><td> N / A </td><td> 200 ml </td></tr><tr><td> Trapeadores Buffer </td><td> N / A </td><td> N / A </td><td> 5 ml </td></tr><tr><td> Fosfato de sodio </td><td> Sigma </td><td> S-9638 </td><td> 0,168 g </td></tr><tr><td> El piruvato de sodio </td><td> Sigma </td><td> P5280 </td><td> 0,22 g </td></tr><tr><td> EDTA de sodio salt </td><td> Sigma </td><td> ED2SS </td><td> 0,0074 g </td></tr><tr><td> Glucosa </td><td> Sigma </td><td> G7528 </td><td> 0,901 g </td></tr><tr><td> Albúmina, bovina </td><td> USB </td><td> 10856 </td><td> 10 g </td></tr><tr><td> El agua filtrada estéril </td><td> N / A </td><td> N / A </td><td> Llevar a 1 L </td></tr><tr><td> Ajustar el pH de 7,4 a 37 ° C, luego de filtro estéril en botellas autoclave y se almacena a 4 º C. </td></tr></tbody></table> Tabla 1. Reactivos específicos utilizados. Almacene todos a 4 ° C.

measurement of cytosolic ca2+ in isolated contractile lymphatics

Los vasos linfáticos forman un sistema de transporte multifuncional que mantiene la homeostasis de fluidos, proporciona los lípidos de la circulación central, y actúa como un sistema de vigilancia para los antígenos potencialmente peligrosos, la optimización de inmunidad de la mucosa y la respuesta inmune adaptativa 1. Linfático está formado por el líquido intersticial que entra ciegos composición linfáticos iniciales, y luego se transporta en contra de un gradiente de presión en los vasos linfáticos más grandes de recolección. Cada linfático recolección se compone de una serie de segmentos llamados linfangiones, separados por válvulas bicúspides que evitar el reflujo. Cada lymphangion posee un ciclo de contracción que impulsa la linfa en contra de un gradiente de presión hacia la circulación central 2. Este patrón de contracción fásica es análoga a la del ciclo cardíaco, con fases sistólica y diastólica, y con una contracción del 4 de baja frecuencia. Además, el músculo liso linfático genera tono y muestra la constricción y la dilatación miogénica in respuesta a los aumentos y disminuciones en la presión luminal, respectivamente 5. Un híbrido de los mecanismos moleculares que apoyan tanto la contractilidad fásica y tónica de los linfáticos Así, se proponen. Contracción del músculo liso está regulada por la citosólica de Ca 2 + de concentración ([Ca 2 +] i), además de la sensibilidad a Ca 2 +, de los elementos contráctiles en respuesta a cambios en el entorno que rodea la célula 6. [Ca 2 +] i se determina por la combinación del movimiento de la Ca 2 + a través de la membrana plasmática del ligando o voltaje de Ca2 + y los canales de la liberación y absorción de Ca 2 + en las tiendas internas. Citosólica de Ca 2 + se une a la calmodulina y activa las enzimas tales como la cadena ligera de la miosina (MLC) quinasa (MLCK), que a su vez fosforila MLC que conduce a la actina-miosina mediada por la contracción 8. Sin embargo, la sensibilidad de esta vía a Ca <sup&gt; 2 + puede ser regulada por la fosfatasa MLC (PCML) 9. PCML actividad está regulada por la Rho quinasa (ROCK) y la fosfatasa miosina inhibidor de la proteína CPI-17. A continuación, presentamos un método para evaluar los cambios en la [Ca 2 +] i en el tiempo en los vasos linfáticos aislados, perfundidos con el fin de estudiar Ca 2 +-dependiente y Ca 2 +-sensibilizar a los mecanismos de contracción del músculo liso linfático. El uso aislado linfáticos mesentéricos de ratas se estudió la recogida estirar los cambios inducidos en el [Ca 2 +] i y la actividad contráctil. El modelo linfático aislado ofrece la ventaja de que la presión, el flujo y la composición química de la solución del baño puede ser muy controlado. [Ca 2 +] i fue determinada por la carga de vasos linfáticos con el radiométrica, Ca 2 +-unión del colorante Fura-2. Estos estudios proporcionan un nuevo enfoque al problema más amplio de estudio de los diferentes mecanismos moleculares que regulan fásicacontracciones tónico contra la constricción del músculo liso linfático.

Watch this Scientific Journal Video about Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics at JoVE.com

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Se introduce un enfoque para evaluar la citosólica de Ca<sup&gt; 2 +</sup&gt; Concentración en los vasos linfáticos aislados para estudiar Ca<sup&gt; 2 +</sup&gt;-Dependiente y Ca<sup&gt; 2 +</sup&gt; Sensibilizante mecanismos de contracción del músculo liso linfático.

Medición de la citosólica de Ca 2 + En aislados linfáticos contráctil

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

measurement-of-cytosolic-ca2-in-isolated-contractile-lymphatics

Research

JoVE Journal

Immunology and Infection

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

13.5K vistas.  Louisiana State University Health Sciences Center. Se introduce un enfoque para evaluar la citosólica de Ca 2 + Concentración en los vasos linfáticos aislados para estudiar Ca 2 +-Dependiente y Ca 2 + Sensibilizante mecanismos de contracción del músculo liso linfático.

Video: Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

La medición de flujos rápidos de calcio en los cardiomiocitos

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Investigación

Biología

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

Aislamiento, cultivo y caracterización funcional de ratón adulto Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Análisis en tiempo real del calcio y la contractilidad en vasos linfáticos aislados

Real-time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i).
This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]i&#160;and diameter measurements continuously in real time.
The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.

Autor destacado: Métodos innovadores en la investigación del linfedema y la hipertensión

This protocol describes a method to simultaneously measure cytosolic, free calcium [Ca2+]i and vessel diameter in contracting lymph vessels in real time and then calculate absolute Ca2+ concentrations as well as contractility/rhythmicity parameters. This protocol can be used to study Ca2+ and contractile dynamics across a variety of experimental conditions.

Evaluación en tiempo real de la contractilidad absoluta, citosólica, libre de Ca2+ y correspondiente en vasos linfáticos aislados y presurizados

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical ...

Inmunología e Infección

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Las mediciones del calcio citosólico en las células epiteliales renales mediante citometría de flujo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Contractilidad Mediciones en músculos papilares aislados para la Investigación de Cardíaca inotropía en ratones

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

De imagen confocal de medición de las fluctuaciones de calcio en el retículo sarcoplásmico de células musculares lisas cultivadas Uso de FRET a base de

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial malfunction in the diseased heart remains to be fully clarified. Recent compelling evidence demonstrates that changes in the myofilament Ca2+ sensitivity affect intracellular Ca2+ homeostasis and ion channel activities in cardiac myocytes, the essential mechanisms responsible for the cardiac action potential and contraction in healthy and diseased hearts. Indeed, activities of ion channels and transporters underlying cardiac action potentials (e.g., Na+, Ca2+ and K+ channels and the Na+-Ca2+ exchanger) and intracellular Ca2+ handling proteins (e.g., ryanodine receptors and Ca2+-ATPase in sarcoplasmic reticulum (SERCA2a) or phospholamban and its phosphorylation) are conventionally measured to evaluate the fundamental mechanisms of cardiac excitation-contraction (E-C) coupling. Both electrical activities in the membrane and intracellular Ca2+ changes are the trigger signals of E-C coupling, whereas myofilament is the functional unit of contraction and relaxation, and myofilament Ca2+ sensitivity is imperative in the implementation of myofibril performance. Nevertheless, few studies incorporate myofilament Ca2+ sensitivity into the functional analysis of the myocardium unless it is the focus of the study. Here, we describe a protocol that measures sarcomere shortening/re-lengthening and the intracellular Ca2+ level using Fura-2 AM (ratiometric detection) and evaluate the changes of myofilament Ca2+ sensitivity in cardiac myocytes from rat hearts. The main aim is to emphasize that myofilament Ca2+ sensitivity should be taken into consideration in E-C coupling for mechanistic analysis. Comprehensive investigation of ion channels, ion transporters, intracellular Ca2+ handling, and myofilament Ca2+ sensitivity that underlie myocyte contractility in healthy and diseased hearts will provide valuable information for designing more effective strategies of translational and therapeutic value.

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

Evaluación de los miofilamentos Ca<sup&gt; 2+</sup&gt; Sensibilidad cardíaca subyacente excitación-contracción de acoplamiento

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Aislamiento de myofibrils de biopsias de músculo esquelético y determinación de la función contráctil con un transductor de fuerza de resolución Nano-Newton

Medición de la citosólica de Ca^{2 +} En aislados linfáticos contráctil

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