Figure 1 - uploaded by Eduardo Ríos
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Absorption spectra of phenol red and ApIII at various pH. The figure graphs absorption spectra of intracellular solutions containing 2 mM phenol red (red trace) or 0.88 mM ApIII measured in a 0.1-cm cuvette. Note lack of sensitivity of ApIII absorption to pH.
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 and [Ca 2 ](t). Pulse...](profile/Eduardo-Rios/publication/8374330/figure/fig3/AS:394387814141952@1471040790312/Simultaneously-measured-H-t-and-Ca-2-t-Pulse-patterns-are-represented_Q320.jpg)
![Figure 6. Two estimates of Ca 2 release flux. (A) d[H ]/dt derived from...](profile/Eduardo-Rios/publication/8374330/figure/fig5/AS:668905433481222@1536490891563/Two-estimates-of-Ca-2-release-flux-A-dH-dt-derived-from-H-t-in-Fig-5-B_Q320.jpg)
In skeletal muscle, the waveform of Ca(2+) release under clamp depolarization exhibits an early peak. Its decay reflects an inactivation, which locally corresponds to the termination of Ca(2+) sparks, and is crucial for rapid control. In cardiac muscle, both the frequency of spontaneous sparks (i.e., their activation) and their termination appear t...
Citations
... Like transmembrane voltage, the Ca 2+ current through SR RyRs that results from TT membrane depolarization cannot be measured directly. Instead, it can be calculated (when other sources are negligible) from the time course of the increase in Ca 2+ concentration in the myoplasm derived from an indicator dye (Baylor et al., 1983;Melzer et al., 1987;Pizarro and Ríos, 2004). The calculation involves estimates of the amounts of Ca 2+ bound to intracellular sites and removed by active transport. ...
Calcium ions control multiple physiological functions by binding to extracellular and intracellular targets. One of the best-studied Ca ²⁺ -dependent functions is contraction of smooth and striated muscle tissue, which results from Ca ²⁺ ligation to calmodulin and troponin C, respectively. Ca ²⁺ signaling typically involves flux of the ion across membranes via specifically gated channel proteins. Because calcium ions are charged, they possess the ability to generate changes in the respective transmembrane voltage. Ca ²⁺ -dependent voltage alterations of the surface membrane are easily measured using microelectrodes. A well-known example is the characteristic plateau phase of the action potential in cardiac ventricular cells that results from the opening of voltage-gated L-type Ca ²⁺ channels. Ca ²⁺ ions are also released from intracellular storage compartments in many cells, but these membranes are not accessible to direct voltage recording with microelectrodes. In muscle, for example, release of Ca ²⁺ from the sarcoplasmic reticulum (SR) to the myoplasm constitutes a flux that is considerably larger than the entry flux from the extracellular space. Whether this flux is accompanied by a voltage change across the SR membrane is an obvious question of mechanistic importance and has been the subject of many investigations. Because the tiny spaces enclosed by the SR membrane are inaccessible to microelectrodes, alternative methods have to be applied. In a study by Sanchez et al. (2018. J. Gen. Physiol.https://doi.org/10.1085/201812035 ) in this issue, modern confocal light microscopy and genetically encoded voltage probes targeted to the SR were applied in a new approach to search for changes in the membrane potential of the SR during Ca ²⁺ release.
... Because the response was very similar for the two probes, pH sensitivity was not a consequence of the mutations. A cytosolic pH change has been well documented in frog muscle fibers loaded with EGTA, where it was used to measure and quantify the EC coupling-activated cytosolic Ca 2+ transients using the EGTA-phenol red method (Pape et al., 1995(Pape et al., , 1998Pizarro and Ríos, 2004). Under conditions of voltage-clamp stimulation similar to the ones used here, Pizarro and Ríos (2004) reported maximum cytosolic levels of increase in [H + ] of 0.2-0.3 ...
... A cytosolic pH change has been well documented in frog muscle fibers loaded with EGTA, where it was used to measure and quantify the EC coupling-activated cytosolic Ca 2+ transients using the EGTA-phenol red method (Pape et al., 1995(Pape et al., , 1998Pizarro and Ríos, 2004). Under conditions of voltage-clamp stimulation similar to the ones used here, Pizarro and Ríos (2004) reported maximum cytosolic levels of increase in [H + ] of 0.2-0.3 µM, which would translate into a change of 0.6-0.8 ...
Ion channel activity in the plasma membrane of living cells generates voltage changes that are critical for numerous biological functions. The membrane of the endoplasmic/sarcoplasmic reticulum (ER/SR) is also endowed with ion channels, but whether changes in its voltage occur during cellular activity has remained ambiguous. This issue is critical for cell functions that depend on a Ca2+ flux across the reticulum membrane. This is the case for contraction of striated muscle, which is triggered by opening of ryanodine receptor Ca2+ release channels in the SR membrane in response to depolarization of the transverse invaginations of the plasma membrane (the t-tubules). Here, we use targeted expression of voltage-sensitive fluorescence resonance energy transfer (FRET) probes of the Mermaid family in differentiated muscle fibers to determine whether changes in SR membrane voltage occur during depolarization-contraction coupling. In the absence of an SR targeting sequence, FRET signals from probes present in the t-tubule membrane allow calibration of the voltage sensitivity and amplitude of the response to voltage-clamp pulses. Successful SR targeting of the probes was achieved using an N-terminal domain of triadin, which completely eliminates voltage-clamp-activated FRET signals from the t-tubule membrane of transfected fibers. In fibers expressing SR-targeted Mermaid probes, activation of SR Ca2+ release in the presence of intracellular ethyleneglycol-bis(β-amino-ethyl ether)-N,N,N',N'-tetra acetic acid (EGTA) results in an accompanying FRET signal. We find that this signal results from pH sensitivity of the probe, which detects cytosolic acidification because of the release of protons upon Ca2+ binding to EGTA. When EGTA is substituted with either 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or the contraction blocker N-benzyl-p-toluene sulfonamide, we find no indication of a substantial change in the FRET response caused by a voltage change. These results suggest that the ryanodine receptor-mediated SR Ca2+ efflux is well balanced by concomitant counterion currents across the SR membrane.
... El MTT incubado sin células también generó absorción entre 500 y 600 nm pero con una intensidad menor. Esta absorción se debe al rojo de fenol [16] presente en el medio de cultivo, el cual deja residuos en los pozos de la placa tras ser decantado. ...
La espectroscopia UV-Vis es una herramienta fundamental en el estudio de los efectos biológicos de las sustancias
químicas. Ésta, es la herramienta más utilizada en la evaluación de los efectos tóxicos o proliferativos de las sustancias
químicas en cultivos celulares, lo que es de amplio interés en los estudios preclínicos para el desarrollo de nuevos
fármacos para tratar el cáncer. La espectroscopia UV-Vis es una técnica robusta, fácil, económica y rápida que presenta
múltiples ventajas en la evaluación de la viabilidad celular in vitro. El presente trabajo expone los fundamentos de la
espectroscopia UV-Vis en el estudio de la viabilidad celular y presenta la evaluación del efecto de algunas sustancias y
extractos de plantas en la viabilidad de cultivos celulares de cáncer de mama.
... The values obtained in the fit do not necessarily reflect the actual properties of each Ca 2+ binding site but the success of the removal model as a whole to reproduce the decay of the [Ca 2+ ] myo transients after the end of voltage clamp pulses of various amplitudes and durations. The reliability of the method has been shown by Pizarro and Ríos (2004). The waveform obtained, loosely referred as Ca 2+ release flux, is equal to the rate of change in total [Ca 2+ ] myo (free plus bound) plus the rate at which Ca 2+ is pumped back to the SR. ...
... Not only was the peak release drastically reduced but the rate of decay after the peak was also slowed down. This has been previously observed and interpreted as a reduction in the rate of Ca 2+ dependent inactivation (Schneider and Simon 1988;Pizarro and Ríos 2004;Olivera and Pizarro 2010). ...
[Ca²⁺] transients inside the sarcoplasmic reticulum (SR) were recorded in frog skeletal muscle twitch fibers under voltage clamp using the low affinity indicator Mag Fluo 4 (loaded in its AM form) with the purpose of studying the effect on Ca²⁺ release of extrinsic Ca²⁺ buffers (i.e. BAPTA) added at high concentration to the myoplasm. When the extrinsic Ca²⁺ buffer is added to the myoplasm, part of the released Ca²⁺ binds to it, reducing the Ca²⁺ signal reported by a myoplasmic indicator. This, in turn, hinders the quantification of the amount of Ca²⁺ released. Monitoring release by measuring [Ca²⁺] inside the SR avoids this problem. The application of extrinsic buffers at high concentration reduced the resting [Ca²⁺] in the SR ([Ca²⁺]SR) continuously from a starting value close to 400 μM reaching the range of 100 μM in about half an hour. The effect of reducing resting [Ca²⁺]SR on the Ca²⁺ permeability of the SR activated by voltage clamp depolarization to 0 mV was studied in cells where the myoplasmic [Ca²⁺] ([Ca²⁺]myo) transients were simultaneously recorded with Rhod2. The Ca²⁺ release flux was calculated from [Ca²⁺]myo and divided by [Ca²⁺]SR to obtain the permeability. Peak permeability was significantly reduced, from 0.026 ± 0.005 ms⁻¹ at resting [Ca²⁺]SR = 372 ± 5 μM to 0.021 ± 0.004 ms⁻¹ at resting [Ca²⁺]SR = 120 ± 16 μM (n = 4, p = 0.03). The time averaged permeability was not significantly changed (0.009 ± 0.003 and 0.010 ± 0.003 ms⁻¹, at the higher and lower [Ca²⁺]SR respectively). Once the cells were equilibrated with the high buffer intracellular solution, the change in [Ca²⁺]SR (Δ[Ca²⁺]SR) in response to voltage clamp depolarization (0 mV, 200 ms) in 20 mM BAPTA was significantly lower (Δ[Ca²⁺]SR = 30.2 ± 3.5 μM from resting [Ca²⁺]SR = 88.8 ± 13.6 μM, n = 5) than in 40 mM EGTA (Δ[Ca²⁺]SR = 72.2 ± 10.4 μM from resting [Ca²⁺]SR = 98.2 ± 15.6 μM, n = 4) suggesting that a Ca²⁺ activated component of release was suppressed by BAPTA.
... The disparate interpretations of the loss or blunting of flux peak emerged from different methods to evaluate the flux magnitude, which resulted in strikingly different scalings of a similar waveform (Fig. 6 B, blue and red traces). Although the disagreement was never truly solved, Pizarro and Ríos (2004) tried to reconcile the conclusions by applying both methods (the removal analysis of Melzer et al., 1987, used in the Schneider laboratory, and the EGTA-phenol red method of Chandler and colleagues) simultaneously to the same frog cells. Their conclusion essentially split the difference, finding buffer-induced reductions in both CICR and CDI. ...
Ryanodine-sensitive intracellular Ca2+ channels (RyRs) open upon binding Ca2+ at cytosolic-facing sites. This results in concerted, self-reinforcing opening of RyRs clustered in specialized regions on the membranes of Ca2+ storage organelles (endoplasmic reticulum and sarcoplasmic reticulum), a process that produces Ca2+-induced Ca2+ release (CICR). The process is optimized to achieve large but brief and localized increases in cytosolic Ca2+ concentration, a feature now believed to be critical for encoding the multiplicity of signals conveyed by this ion. In this paper, I trace the path of research that led to a consensus on the physiological significance of CICR in skeletal muscle, beginning with its discovery. I focus on the approaches that were developed to quantify the contribution of CICR to the Ca2+ increase that results in contraction, as opposed to the flux activated directly by membrane depolarization (depolarization-induced Ca2+ release [DICR]). Although the emerging consensus is that CICR plays an important role alongside DICR in most taxa, its contribution in most mammalian muscles appears to be limited to embryogenesis. Finally, I survey the relevance of CICR, confirmed or plausible, to pathogenesis as well as the multiple questions about activation of release channels that remain unanswered after 50 years.
... The values obtained in the fit do not necessarily reflect the actual properties of each Ca 2? binding site but rather the success of the removal model as a whole to reproduce the decay of the [Ca 2? ] transients after the end of voltage clamp pulses of various amplitudes and durations. The reliability of the method has been shown by Pizarro and Ríos (2004). The waveform obtained, loosely referred as Ca 2? release flux, is equal to the rate of change in total myoplasmic [Ca 2? ] plus the rate at which Ca 2? is pumped back to the SR. ...
Raising the intracellular [Ca(2+)] ([Ca(2+)]i) was previously found to produce uncoupling between the electrical depolarization of the transverse tubules and contraction in skinned muscle fibers. Here we study the effect of elevated [Ca(2+)]i in voltage clamped cut fibers of frog skeletal muscle to establish how the charge movement, a measure of the activation of the dihydropyridine receptors (DHPR)-voltage sensors, and Ca(2+) release, a consequence of the opening of the ryanodine receptor (RyR)-release channels, were affected. [Ca(2+)]i was raised by various procedures (pharmacological release from the sarcoplasmic reticulum, application of high [Ca(2+)]i intracellular solution, permeabilization of the plasma membrane by a Ca(2+) ionophore) all of which produced impairment of excitation-contraction coupling. The charge movement was reduced from 20.2 ± 1.24 to 9.9 ± 0.94 nC/μF meanwhile the Ca(2+) release flux was reduced from 13.5 + 0.7 to 2.2 ± 0.3 μM/ms (n = 33). This suggests that a significant fraction of the DHPRs that remained functional, could not activate RyRs, and were therefore presumably disconnected. These results are broadly consistent with the original reports in skinned fibers. Uncoupling was prevented by the addition to the intracellular solution of the protease inhibitor leupeptin. In approximately 40 % of the uncoupled cells we observed that the [Ca(2+)]i transient continued to rise after the voltage clamp pulse was turned off. This loss of control by membrane voltage suggests that the uncoupled release channels might have another mechanism of activation, likely by Ca(2+).
... (The estimate of calcium in the SR was 77% for EDL muscle, largely composed of fast-twitch fibers, and an even greater fraction in soleus, which lacks calciumbinding parvalbumin in the cytosol.) For the frog, these numbers translate to 2.5 mmoles/kg of myoplasmic water, in substantial agreement with the estimates of total releasable calcium in cut fibers under voltage clamp (Pape et al., 1995;Pizarro and Ríos, 2004). visibly skewed, or worse than the other, suggesting that numeric analysis of errors will not help choose between these alternative possibilities. ...
The calcium ion is the most universal and widely studied vector of chemical signals in living cells. Its presence and dynamic distribution in diverse organelles are associated with various functions, which include controlling pro- tein expression and activity in response to functional demands. Calcium’s role was rst investigated in striated muscles, where it regulates contractile force. Muscles contain large amounts of calcium, most of which is selec- tively bound inside speci c organelles. Its homeostasis and dynamics emerge from the precise interaction of various proteins, including channels of the plasmalemma and intracellular organelles, ion pumps, and both target and storage proteins. The large magnitude of the cyclic movements of calcium between the SR and cytosol re- quires an intra-SR calcium-binding molecule of large capacity and low af nity. These properties are found in calsequestrin, viewed not just as the main SR calcium- binding protein but also as a modulator of the pathway for calcium release. Cytosolic proteins, including tropo- nin C and parvalbumin, as well as mitochondria, can also store substantial quantities of calcium.
Although great strides have been made in developing various reporters for measuring the concentration of the calcium ion and its dynamics in cytosol and organ- elles, the measurement of total steady calcium content in tissues has been, well, static, limited by the dif culties of the sole technique in wide use, namely atomic ab- sorption spectroscopy (AAS; also known as ame pho- tometry), which is done in supernatant solution after sample homogenization, denaturation, and removal of protein. Lamboley et al. (2015) now present a welcome alternative to AAS.
... (The estimate of calcium in the SR was 77% for EDL muscle, largely composed of fast-twitch fibers, and an even greater fraction in soleus, which lacks calciumbinding parvalbumin in the cytosol.) For the frog, these numbers translate to 2.5 mmoles/kg of myoplasmic water, in substantial agreement with the estimates of total releasable calcium in cut fibers under voltage clamp (Pape et al., 1995;Pizarro and Ríos, 2004). visibly skewed, or worse than the other, suggesting that numeric analysis of errors will not help choose between these alternative possibilities. ...
The calcium ion is the most universal and widely studied vector of chemical signals in living cells. Its presence and dynamic distribution in diverse organelles are associated with various functions, which include controlling protein expression and activity in response to functional demands. Calcium's role was first investigated in striated muscles, where it regulates contractile force. Muscles contain large amounts of calcium, most of which is selectively bound inside specific organelles. Its homeostasis and dynamics emerge from the precise interaction of various proteins, including channels of the plasmalemma and intracellular organelles, ion pumps, and both target and storage proteins. The large magnitude of the cyclic movements of calcium between the SR and cytosol requires an intra-SR calcium-binding molecule of large capacity and low affinity. These properties are found in calsequestrin, viewed not just as the main SR calcium-binding protein but also as a modulator of the pathway for calcium release. Cytosolic proteins, including tropo-nin C and parvalbumin, as well as mitochondria, can also store substantial quantities of calcium. Although great strides have been made in developing various reporters for measuring the concentration of the calcium ion and its dynamics in cytosol and organ-elles, the measurement of total steady calcium content in tissues has been, well, static, limited by the difficulties of the sole technique in wide use, namely atomic absorption spectroscopy (AAS; also known as flame pho-tometry), which is done in supernatant solution after sample homogenization, denaturation, and removal of protein. Lamboley et al. (2015) now present a welcome alternative to AAS. The new method relies on fluorescence of BAPTA The first virtue of their new method is its simple rationale. It relies on the major shift in UV absorption spectrum of the calcium buffer BAPTA upon binding calcium. The authors delineate a technique that involves sample homogenization and removal of solids, and then derives Ca 2+ concentration in the supernatant from the change in absorbance upon removal of the ion from BAPTA after the addition of EGTA. A salient design feature is the chosen concentration of BAPTA, calculated Correspondence to Carlo Manno: c a r l o s _ m a n n o @ r u s h. e d u to bind essentially all calcium, while avoiding any excess that would reduce the dynamic range of the signal and hence the sensitivity of the method. The paper is largely concerned with validating several assumptions that make the math simple and the process straightforward. Thus, the authors demonstrate that BAPTA ultimately binds all calcium present in the sample so that other ions do not affect or interfere with the amount of calcium-bound BAPTA, and that neither the intrinsic absorbance of the sample nor the binding of proteins to BAPTA interferes with the assay. Given the general adherence of the results to these assumptions, the initial set of measures from various muscles of three animal species was narrowly distributed and allow for two-or three-figure precision in the averages—a major advance. As for the actual calcium content, the numbers for mouse extensor digitorum longus (EDL; 2.71 ± 0.09; average ± SEM) and soleus (2.62 ± 0.11), in units of mil-limole per kilogram of muscle wet weight, are of most interest for current studies. The corresponding numbers for rat are about half of those for mice. These values are in substantial agreement with earlier results obtained with AAS in rats (Everts et al., 1993; Gissel and Clausen, 1999) but >50% greater than the earlier measurements in various mouse leg muscles. Most calcium is in the SR
... (The estimate of calcium in the SR was 77% for EDL muscle, largely composed of fast-twitch fibers, and an even greater fraction in soleus, which lacks calciumbinding parvalbumin in the cytosol.) For the frog, these numbers translate to 2.5 mmoles/kg of myoplasmic water, in substantial agreement with the estimates of total releasable calcium in cut fibers under voltage clamp (Pape et al., 1995;Pizarro and Ríos, 2004). visibly skewed, or worse than the other, suggesting that numeric analysis of errors will not help choose between these alternative possibilities. ...
... Por ser breve la despolarización (el potencial de acción), la repolarización, con el consiguiente retorno de los sensores de voltaje a su configuración de reposo, cierra rápidamente los canales RyR (el mecanismo llamado " desactivación " ). Sin embargo, desde tiempo atrás se sabe que existe un mecanismo coadyuvante, llamado inactivación dependiente de calcio, IDC, por el cual el mismo calcio que atraviesa un canal se une a sitios en la faz citosólica del mismo canal (o canales próximos) y causa su cierre [41]. Una prueba clásica de la presencia de este mecanismo se hace agregando al medio citosólico el tampón de calcio BAPTA. ...
Este artículo examina la evolución del concepto de cuplón, unidad funcional del acoplamiento contráctil, originado hace más de quince años con los colegas Gonzalo Pizarro y Michael Stern. Este examen nos permitirá poner al día avances en el estudio del acoplamiento entre la excitación de origen nervioso y la contracción muscular. Como se verá, la investigación en el tema ha tenido recientemente un claro viraje hacia la búsqueda de formas de aplicación de los conceptos básicos en la prevención y el tratamiento de varias enfermedades. A su vez, el estudio de mutaciones hereditarias en las proteínas del cuplón permitió nuevos avances en la comprensión de las interrogantes más básicas (cuáles son sus funciones y sus interacciones supramoleculares) en una feliz y bastante inesperada potenciación mutua entre enfoques muy diferentes de la realidad. Este artículo sigue un desarrollo iterativo, describiendo primero los aspectos básicos del tema, pasando luego a los estudios aplicados, y a través de ellos volviendo a cuestiones fundamentales, ahora en pleno proceso de esclarecimiento.
