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Stephen M. Baylor - One of the best experts on this subject based on the ideXlab platform.
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The Amplitude and Time Course of the Myoplasmic Free [Ca 2+] Transient in Fast-twitch Fibers of Mouse Muscle
2013Co-Authors: Stephen Hollingworth, Mingdi Zhao, Stephen M. BaylorAbstract:ABSTRACT Bundles of 10-100 fibers were dissected from the extensor digitorum longus muscle of mouse, mounted in an apparatus for optical recording, and stretched to long sarcomere length (-> 3.6 p~m). One fiber within the bundle was microinjected with Furaptra, a fluorescent indicator that responds rapidly to changes in myoplasmic free [Ca 2+] (A[Ca2+]). Twitches and brief tetani were initiated by external stimulation. At myoplasmic Furaptra concentrations of ~0.1 mM, the indicator's fluorescence signal during fiber activity (AF/F) was well resolved. AF/F was converted to A [Ca 2+] under the assumption that Furaptra's myoplasmic dissociation constant for Ca ~+ is 98 ~M at 16 ~ and 109 p~M at 28 ~ At 16 ~ the peak amplitude ofA[Ca ~+] during a twitch was 17.8-+ 0.4 ~M (-+SEM; n = 8) and the half-width of A[Ca 2+] was 4.6 +- 0.3 ms. At 28 ~ the peak and half-width values were 22.1- 1.8 ~zM and 2.0-+ 0.1 ms, respectively (n = 4). During a brief high-frequency tetanus, individual peaks of A [Ca 2+] were also well resolved and reached approximately the same amplitude that resulted from a single shock; the initial decays of A[Ca z+] from peak slowed substantially during the tetanus. For a single twitch at 160C, the amplitude of A [Ca 2+] in fast-twitch fibers of mouse is not significantly different from that recently measured in fasttwitch fibers of frog (16.5 _+ 0.9 IxM; Zhao, M., S. Hollingworth, and S.M. Baylor. 1996. Biophys.J. 70:896-916); in contrast, the half-width of A[Ca 2+] is surprisingly brief in mouse fibers, only about half that measured in frog (9.6 + 0.6 ms). The estimated peak rate at which Ca 2+ is released from the sarcoplasmic reticulum in response to an action potential is also similar in mouse and frog, 140-150 I~M/ms (16~ KEY WORDS: calcium transients 9 mammalian muscle 9 fast-twitch fibers 9 Furaptra 9 calcium indicator
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comparison of myoplasmic calcium movements during excitation contraction coupling in frog twitch and mouse fast twitch muscle fibers
The Journal of General Physiology, 2013Co-Authors: Stephen Hollingworth, Stephen M. BaylorAbstract:Single twitch fibers from frog leg muscles were isolated by dissection and micro-injected with Furaptra, a rapidly responding fluorescent Ca2+ indicator. Indicator resting fluorescence (FR) and the change evoked by an action potential (ΔF) were measured at long sarcomere length (16°C); ΔF/FR was scaled to units of ΔfCaD, the change in fraction of the indicator in the Ca2+-bound form. ΔfCaD was simulated with a multicompartment model of the underlying myoplasmic Ca2+ movements, and the results were compared with previous measurements and analyses in mouse fast-twitch fibers. In frog fibers, sarcoplasmic reticulum (SR) Ca2+ release evoked by an action potential appears to be the sum of two components. The time course of the first component is similar to that of the entire Ca2+ release waveform in mouse fibers, whereas that of the second component is severalfold slower; the fractional release amounts are ∼0.8 (first component) and ∼0.2 (second component). Similar results were obtained in frog simulations with a modified model that permitted competition between Mg2+ and Ca2+ for occupancy of the regulatory sites on troponin. An anatomical basis for two release components in frog fibers is the presence of both junctional and parajunctional SR Ca2+ release channels (ryanodine receptors [RyRs]), whereas mouse fibers (usually) have only junctional RyRs. Also, frog fibers have two RyR isoforms, RyRα and RyRβ, whereas the mouse fibers (usually) have only one, RyR1. Our simulations suggest that the second release component in frog fibers functions to supply extra Ca2+ to activate troponin, which, in mouse fibers, is not needed because of the more favorable location of their triadic junctions (near the middle of the thin filament). We speculate that, in general, parajunctional RyRs permit increased myofilament activation in fibers whose triadic junctions are located at the z-line.
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measurement and simulation of myoplasmic calcium transients in mouse slow twitch muscle fibres
The Journal of Physiology, 2012Co-Authors: Stephen Hollingworth, Michele M Kim, Stephen M. BaylorAbstract:Non-technical summary In voluntary muscle cells, the change in the intracellular concentration of calcium ions (Δ[Ca2+]) controls the contractile cycle by controlling the binding of Ca2+ to the ‘regulatory’ sites on the troponin molecules attached to the myofilaments. Δ[Ca2+] is difficult to measure under physiological conditions, as indicated by the discrepancies in results from recent studies of Δ[Ca2+] on mouse slow-twitch muscle fibres. Here we examine some of the reasons underlying these discrepancies and argue that measurements made with membrane-impermeant Ca2+ indicators that are micro-injected into intact fibres are more accurate than those made with indicators introduced into enzyme-dissociated fibres by diffusion (“AM loading”). Using a computational model, we have analysed the Δ[Ca2+] measurements to estimate the kinetic rate constants that govern the reaction of Ca2+ with the troponin regulatory sites. To our knowledge, this is the first time such estimates have been deduced from physiological measurements in living slow-twitch muscle fibres. Abstract Bundles of intact fibres from soleus muscles of adult mice were isolated by dissection and one fibre within a bundle was micro-injected with either Furaptra or mag-fluo-4, two low-affinity rapidly responding Ca2+ indicators. Fibres were activated by action potentials to elicit changes in indicator fluorescence (ΔF), a monitor of the myoplasmic free Ca2+ transient (Δ[Ca2+]), and changes in fibre tension. All injected fibres appeared to be slow-twitch (type I) fibres as inferred from the time course of their tension responses. The full-duration at half-maximum (FDHM) of ΔF was found to be essentially identical with the two indicators; the mean value was 8.4 ± 0.3 ms (±SEM) at 16°C and 5.1 ± 0.3 ms at 22°C. The value at 22°C is about one-third that reported previously in enzyme-dissociated slow-twitch fibres that had been AM-loaded with mag-fluo-4: 12.4 ± 0.8 ms and 17.2 ± 1.7 ms. We attribute the larger FDHM in enzyme-dissociated fibres either to an alteration of fibre properties due to the enzyme treatment or to some error in the measurement of ΔF associated with AM loading. ΔF in intact fibres was simulated with a multi-compartment reaction-diffusion model that permitted estimation of the amount and time course of Ca2+ release from the sarcoplasmic reticulum (SR), the binding and diffusion of Ca2+ in the myoplasm, the re-uptake of Ca2+ by the SR Ca2+ pump, and Δ[Ca2+] itself. In response to one action potential at 16°C, the following estimates were obtained: 107 μm for the amount of Ca2+ release; 1.7 ms for the FDHM of the release flux; 7.6 μm and 4.9 ms for the peak and FDHM of spatially averaged Δ[Ca2+]. With five action potentials at 67 Hz, the estimated amount of Ca2+ release is 186 μm. Two important unknown model parameters are the on- and off-rate constants of the reaction between Ca2+ and the regulatory sites on troponin; values of 0.4 × 108m−1 s−1 and 26 s−1, respectively, were found to be consistent with the ΔF measurements.
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Low-Affinity Ca2+ Indicators Compared in Measurements of Skeletal Muscle Ca2+ Transients
Biophysical journal, 2009Co-Authors: Stephen Hollingworth, Kyle Gee, Stephen M. BaylorAbstract:The low-affinity fluorescent Ca2+ indicators OGB-5N, Fluo-5N, fura-5N, Rhod-5N, and Mag-fluo-4 were evaluated for their ability to accurately track the kinetics of the spatially averaged free Ca2+ transient (Δ[Ca2+]) in skeletal muscle. Frog single fibers were injected with one of the above indicators and, usually, Furaptra (previously shown to rapidly track Δ[Ca2+]). In response to an action potential, the full duration at half-maximum of the indicator's fluorescence change (ΔF) was found to be larger with OGB-5N, Fluo-5N, fura-5N, and Rhod-5N than with Furaptra; thus, these indicators do not track Δ[Ca2+] with kinetic fidelity. In contrast, the ΔF time course of Mag-fluo-4 was identical to Furaptra's; thus, Mag-fluo-4 also yields reliable kinetic information about Δ[Ca2+]. Mag-fluo-4's ΔF has a larger signal/noise ratio than Furaptra's (for similar indicator concentrations), and should thus be more useful for tracking Δ[Ca2+] in small cell volumes. However, because the resting fluorescence of Mag-fluo-4 probably arises largely from indicator that is bound with Mg2+, the amplitude of the Mag-fluo-4 signal, and its calibration in Δ[Ca2+] units, is likely to be more sensitive to variations in [Mg2+] than Furaptra's.
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sarcoplasmic reticulum calcium release compared in slow twitch and fast twitch fibres of mouse muscle
The Journal of Physiology, 2003Co-Authors: Stephen M. Baylor, Stephen HollingworthAbstract:Experiments were carried out to compare the amplitude and time course of Ca2+ release from the sarcoplasmic reticulum (SR) in intact slow-twitch and fast-twitch mouse fibres. Individual fibres within small bundles were injected with Furaptra, a low-affinity, rapidly responding Ca2+ indicator. In response to a single action potential at 16 °C, the peak amplitude and half-duration of the change in myoplasmic free [Ca2+] (Δ[Ca2+]) differed significantly between fibre types (slow-twitch: peak amplitude, 9.4 ± 1.0 μM (mean ± S.E.M.); half-duration, 7.7 ± 0.6 ms; fast-twitch: peak amplitude 18.5 ± 0.5 μM; half-duration, 4.9 ± 0.3 ms). SR Ca2+ release was estimated from Δ[Ca2+] with a computational model that calculated Ca2+ binding to the major myoplasmic Ca2+ buffers (troponin, ATP and parvalbumin); buffer concentrations and reaction rate constants were adjusted to reflect fibre-type differences. In response to an action potential, the total concentration of released Ca2+ (Δ[CaT]) and the peak rate of Ca2+ release ((d/dt)Δ[CaT]) differed about 3-fold between the fibre types (slow-twitch: Δ[CaT], 127 ± 7 μM; (d/dt)Δ[CaT], 70 ± 6 μM ms−1; fast-twitch: Δ[CaT], 346 ± 6 μM; (d/dt)Δ[CaT], 212 ± 4 μM ms−1). In contrast, the half-duration of (d/dt)Δ[CaT] was very similar in the two fibre types (slow-twitch, 1.8 ± 0.1 ms; fast-twitch, 1.6 ± 0.0 ms). When fibres were stimulated with a 5-shock train at 67 Hz, the peaks of (d/dt)Δ[CaT] in response to the second and subsequent shocks were much smaller than that due to the first shock; the later peaks, expressed as a fraction of the amplitude of the first peak, were similar in the two fibre types (slow-twitch, 0.2–0.3; fast-twitch, 0.1–0.3). The results support the conclusion that individual SR Ca2+ release units function similarly in slow-twitch and fast-twitch mammalian fibres.
Masato Konishi - One of the best experts on this subject based on the ideXlab platform.
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Magnesium homeostasis in cardiac myocytes of mg-deficient rats. PloS One 8: e73171
2013Co-Authors: Michiko Tashiro, Hana Inoue, Masato KonishiAbstract:To study possible modulation of Mg2+ transport in low Mg2+ conditions, we fed either a Mg-deficient diet or a Mg-containing diet (control) to Wistar rats for 1–6 weeks. Total Mg concentrations in serum and cardiac ventricular tissues were measured by atomic absorption spectroscopy. Intracellular free Mg2+ concentration ([Mg2+]i) of ventricular myocytes was measured with the fluorescent indicator Furaptra. Mg2+ transport rates, rates of Mg2+ influx and Mg2+ efflux, were estimated from the rates of change in [Mg2+]i during Mg loading/depletion and recovery procedures. In Mg-deficient rats, the serum total Mg concentration (0.2960.026 mM) was significantly lower than in control rats (0.8660.072 mM) after 4–6 weeks of Mg deficiency. However, neither total Mg concentration in ventricular tissues nor [Mg2+]i of ventricular myocytes was significantly different between Mg-deficient rats and control rats. The rates of Mg2+ influx and efflux were not significantly different in both groups. In addition, quantitative RT-PCR revealed that Mg deficiency did not substantially change mRNA expression levels of known Mg2+ channels/transporters (TRPM6, TRPM7, MagT1, SLC41A1 and ACDP2) in heart and kidney tissues. These results suggest that [Mg2+]i as well as the total Mg content of cardiac myocytes, was well maintained even under chronic hypomagnesemia without persistent modulation in function and expression of major Mg2+ channels/ transporters in the heart
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Summary of results obtained from single ventricular myocytes.
2013Co-Authors: Michiko Tashiro, Hana Inoue, Masato KonishiAbstract:Rats (8 weeks old) were fed the control diet or the Mg-deficient diet for 4–6 weeks, and [Mg2+]i was measured with the fluorescent indicator Furaptra in the myocytes isolated from control rats (Control) and Mg-deficient rats (Mg-deficient). Each data represents mean ± SEM from the number of cells indicated in parentheses. The basal level of [Mg2+]i was measured either at 1 mM [Mg2+]o (i.e., Ca2+-free Tyrode’s solution) or at 0.2 mM [Mg2+]o (Mg2+ concentration of Ca2+-free Tyrode’s solution was reduced to 0.2 mM). Mg2+ influx rates were estimated by two different methods: the rates of Mg2+ loading (as shown in Fig. 3) and the rates of Mg2+ recovery after depletion (as shown in Fig. 1). The Mg2+ efflux rate was estimated from the initial rate of decrease in [Mg2+]i in the Mg2+-loaded cells as shown in Fig. 4. There was no significant difference between values obtained from control rats and those obtained from Mg-deficient rats.
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sodium gradient dependent transport of magnesium in rat ventricular myocytes
American Journal of Physiology-cell Physiology, 2000Co-Authors: Michiko Tashiro, Masato KonishiAbstract:Cytoplasmic concentration of Mg2+([Mg2+]i) was measured with a fluorescent indicator Furaptra in ventricular myocytes enzymatically dissociated from rat hearts (25°C). To study Mg2+ transport acros...
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transport of magnesium by two isoforms of the na ca2 exchanger expressed in ccl39 fibroblasts
Pflügers Archiv: European Journal of Physiology, 2000Co-Authors: Michiko Tashiro, Masato Konishi, Takahiro Iwamoto, Munekazu Shigekawa, Satoshi KuriharaAbstract:Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (Δ[Mg2+]i) was monitored with Furaptra under Ca2+-free conditions. In control (vector-transfected) cells, Δ[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. Δ[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of Δ[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 µM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.
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Magnesium homeostasis in cardiac myocytes of Mg-deficient rats.
Public Library of Science (PLoS), 2024Co-Authors: Michiko Tashiro, Hana Inoue, Masato KonishiAbstract:To study possible modulation of Mg(2+) transport in low Mg(2+) conditions, we fed either a Mg-deficient diet or a Mg-containing diet (control) to Wistar rats for 1-6 weeks. Total Mg concentrations in serum and cardiac ventricular tissues were measured by atomic absorption spectroscopy. Intracellular free Mg(2+) concentration ([Mg(2+)]i) of ventricular myocytes was measured with the fluorescent indicator Furaptra. Mg(2+) transport rates, rates of Mg(2+) influx and Mg(2+) efflux, were estimated from the rates of change in [Mg(2+)]i during Mg loading/depletion and recovery procedures. In Mg-deficient rats, the serum total Mg concentration (0.29±0.026 mM) was significantly lower than in control rats (0.86±0.072 mM) after 4-6 weeks of Mg deficiency. However, neither total Mg concentration in ventricular tissues nor [Mg(2+)]i of ventricular myocytes was significantly different between Mg-deficient rats and control rats. The rates of Mg(2+) influx and efflux were not significantly different in both groups. In addition, quantitative RT-PCR revealed that Mg deficiency did not substantially change mRNA expression levels of known Mg(2+) channels/transporters (TRPM6, TRPM7, MagT1, SLC41A1 and ACDP2) in heart and kidney tissues. These results suggest that [Mg(2+)]i as well as the total Mg content of cardiac myocytes, was well maintained even under chronic hypomagnesemia without persistent modulation in function and expression of major Mg(2+) channels/transporters in the heart
Stephen Hollingworth - One of the best experts on this subject based on the ideXlab platform.
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The Amplitude and Time Course of the Myoplasmic Free [Ca 2+] Transient in Fast-twitch Fibers of Mouse Muscle
2013Co-Authors: Stephen Hollingworth, Mingdi Zhao, Stephen M. BaylorAbstract:ABSTRACT Bundles of 10-100 fibers were dissected from the extensor digitorum longus muscle of mouse, mounted in an apparatus for optical recording, and stretched to long sarcomere length (-> 3.6 p~m). One fiber within the bundle was microinjected with Furaptra, a fluorescent indicator that responds rapidly to changes in myoplasmic free [Ca 2+] (A[Ca2+]). Twitches and brief tetani were initiated by external stimulation. At myoplasmic Furaptra concentrations of ~0.1 mM, the indicator's fluorescence signal during fiber activity (AF/F) was well resolved. AF/F was converted to A [Ca 2+] under the assumption that Furaptra's myoplasmic dissociation constant for Ca ~+ is 98 ~M at 16 ~ and 109 p~M at 28 ~ At 16 ~ the peak amplitude ofA[Ca ~+] during a twitch was 17.8-+ 0.4 ~M (-+SEM; n = 8) and the half-width of A[Ca 2+] was 4.6 +- 0.3 ms. At 28 ~ the peak and half-width values were 22.1- 1.8 ~zM and 2.0-+ 0.1 ms, respectively (n = 4). During a brief high-frequency tetanus, individual peaks of A [Ca 2+] were also well resolved and reached approximately the same amplitude that resulted from a single shock; the initial decays of A[Ca z+] from peak slowed substantially during the tetanus. For a single twitch at 160C, the amplitude of A [Ca 2+] in fast-twitch fibers of mouse is not significantly different from that recently measured in fasttwitch fibers of frog (16.5 _+ 0.9 IxM; Zhao, M., S. Hollingworth, and S.M. Baylor. 1996. Biophys.J. 70:896-916); in contrast, the half-width of A[Ca 2+] is surprisingly brief in mouse fibers, only about half that measured in frog (9.6 + 0.6 ms). The estimated peak rate at which Ca 2+ is released from the sarcoplasmic reticulum in response to an action potential is also similar in mouse and frog, 140-150 I~M/ms (16~ KEY WORDS: calcium transients 9 mammalian muscle 9 fast-twitch fibers 9 Furaptra 9 calcium indicator
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comparison of myoplasmic calcium movements during excitation contraction coupling in frog twitch and mouse fast twitch muscle fibers
The Journal of General Physiology, 2013Co-Authors: Stephen Hollingworth, Stephen M. BaylorAbstract:Single twitch fibers from frog leg muscles were isolated by dissection and micro-injected with Furaptra, a rapidly responding fluorescent Ca2+ indicator. Indicator resting fluorescence (FR) and the change evoked by an action potential (ΔF) were measured at long sarcomere length (16°C); ΔF/FR was scaled to units of ΔfCaD, the change in fraction of the indicator in the Ca2+-bound form. ΔfCaD was simulated with a multicompartment model of the underlying myoplasmic Ca2+ movements, and the results were compared with previous measurements and analyses in mouse fast-twitch fibers. In frog fibers, sarcoplasmic reticulum (SR) Ca2+ release evoked by an action potential appears to be the sum of two components. The time course of the first component is similar to that of the entire Ca2+ release waveform in mouse fibers, whereas that of the second component is severalfold slower; the fractional release amounts are ∼0.8 (first component) and ∼0.2 (second component). Similar results were obtained in frog simulations with a modified model that permitted competition between Mg2+ and Ca2+ for occupancy of the regulatory sites on troponin. An anatomical basis for two release components in frog fibers is the presence of both junctional and parajunctional SR Ca2+ release channels (ryanodine receptors [RyRs]), whereas mouse fibers (usually) have only junctional RyRs. Also, frog fibers have two RyR isoforms, RyRα and RyRβ, whereas the mouse fibers (usually) have only one, RyR1. Our simulations suggest that the second release component in frog fibers functions to supply extra Ca2+ to activate troponin, which, in mouse fibers, is not needed because of the more favorable location of their triadic junctions (near the middle of the thin filament). We speculate that, in general, parajunctional RyRs permit increased myofilament activation in fibers whose triadic junctions are located at the z-line.
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measurement and simulation of myoplasmic calcium transients in mouse slow twitch muscle fibres
The Journal of Physiology, 2012Co-Authors: Stephen Hollingworth, Michele M Kim, Stephen M. BaylorAbstract:Non-technical summary In voluntary muscle cells, the change in the intracellular concentration of calcium ions (Δ[Ca2+]) controls the contractile cycle by controlling the binding of Ca2+ to the ‘regulatory’ sites on the troponin molecules attached to the myofilaments. Δ[Ca2+] is difficult to measure under physiological conditions, as indicated by the discrepancies in results from recent studies of Δ[Ca2+] on mouse slow-twitch muscle fibres. Here we examine some of the reasons underlying these discrepancies and argue that measurements made with membrane-impermeant Ca2+ indicators that are micro-injected into intact fibres are more accurate than those made with indicators introduced into enzyme-dissociated fibres by diffusion (“AM loading”). Using a computational model, we have analysed the Δ[Ca2+] measurements to estimate the kinetic rate constants that govern the reaction of Ca2+ with the troponin regulatory sites. To our knowledge, this is the first time such estimates have been deduced from physiological measurements in living slow-twitch muscle fibres. Abstract Bundles of intact fibres from soleus muscles of adult mice were isolated by dissection and one fibre within a bundle was micro-injected with either Furaptra or mag-fluo-4, two low-affinity rapidly responding Ca2+ indicators. Fibres were activated by action potentials to elicit changes in indicator fluorescence (ΔF), a monitor of the myoplasmic free Ca2+ transient (Δ[Ca2+]), and changes in fibre tension. All injected fibres appeared to be slow-twitch (type I) fibres as inferred from the time course of their tension responses. The full-duration at half-maximum (FDHM) of ΔF was found to be essentially identical with the two indicators; the mean value was 8.4 ± 0.3 ms (±SEM) at 16°C and 5.1 ± 0.3 ms at 22°C. The value at 22°C is about one-third that reported previously in enzyme-dissociated slow-twitch fibres that had been AM-loaded with mag-fluo-4: 12.4 ± 0.8 ms and 17.2 ± 1.7 ms. We attribute the larger FDHM in enzyme-dissociated fibres either to an alteration of fibre properties due to the enzyme treatment or to some error in the measurement of ΔF associated with AM loading. ΔF in intact fibres was simulated with a multi-compartment reaction-diffusion model that permitted estimation of the amount and time course of Ca2+ release from the sarcoplasmic reticulum (SR), the binding and diffusion of Ca2+ in the myoplasm, the re-uptake of Ca2+ by the SR Ca2+ pump, and Δ[Ca2+] itself. In response to one action potential at 16°C, the following estimates were obtained: 107 μm for the amount of Ca2+ release; 1.7 ms for the FDHM of the release flux; 7.6 μm and 4.9 ms for the peak and FDHM of spatially averaged Δ[Ca2+]. With five action potentials at 67 Hz, the estimated amount of Ca2+ release is 186 μm. Two important unknown model parameters are the on- and off-rate constants of the reaction between Ca2+ and the regulatory sites on troponin; values of 0.4 × 108m−1 s−1 and 26 s−1, respectively, were found to be consistent with the ΔF measurements.
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Low-Affinity Ca2+ Indicators Compared in Measurements of Skeletal Muscle Ca2+ Transients
Biophysical journal, 2009Co-Authors: Stephen Hollingworth, Kyle Gee, Stephen M. BaylorAbstract:The low-affinity fluorescent Ca2+ indicators OGB-5N, Fluo-5N, fura-5N, Rhod-5N, and Mag-fluo-4 were evaluated for their ability to accurately track the kinetics of the spatially averaged free Ca2+ transient (Δ[Ca2+]) in skeletal muscle. Frog single fibers were injected with one of the above indicators and, usually, Furaptra (previously shown to rapidly track Δ[Ca2+]). In response to an action potential, the full duration at half-maximum of the indicator's fluorescence change (ΔF) was found to be larger with OGB-5N, Fluo-5N, fura-5N, and Rhod-5N than with Furaptra; thus, these indicators do not track Δ[Ca2+] with kinetic fidelity. In contrast, the ΔF time course of Mag-fluo-4 was identical to Furaptra's; thus, Mag-fluo-4 also yields reliable kinetic information about Δ[Ca2+]. Mag-fluo-4's ΔF has a larger signal/noise ratio than Furaptra's (for similar indicator concentrations), and should thus be more useful for tracking Δ[Ca2+] in small cell volumes. However, because the resting fluorescence of Mag-fluo-4 probably arises largely from indicator that is bound with Mg2+, the amplitude of the Mag-fluo-4 signal, and its calibration in Δ[Ca2+] units, is likely to be more sensitive to variations in [Mg2+] than Furaptra's.
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sarcoplasmic reticulum calcium release compared in slow twitch and fast twitch fibres of mouse muscle
The Journal of Physiology, 2003Co-Authors: Stephen M. Baylor, Stephen HollingworthAbstract:Experiments were carried out to compare the amplitude and time course of Ca2+ release from the sarcoplasmic reticulum (SR) in intact slow-twitch and fast-twitch mouse fibres. Individual fibres within small bundles were injected with Furaptra, a low-affinity, rapidly responding Ca2+ indicator. In response to a single action potential at 16 °C, the peak amplitude and half-duration of the change in myoplasmic free [Ca2+] (Δ[Ca2+]) differed significantly between fibre types (slow-twitch: peak amplitude, 9.4 ± 1.0 μM (mean ± S.E.M.); half-duration, 7.7 ± 0.6 ms; fast-twitch: peak amplitude 18.5 ± 0.5 μM; half-duration, 4.9 ± 0.3 ms). SR Ca2+ release was estimated from Δ[Ca2+] with a computational model that calculated Ca2+ binding to the major myoplasmic Ca2+ buffers (troponin, ATP and parvalbumin); buffer concentrations and reaction rate constants were adjusted to reflect fibre-type differences. In response to an action potential, the total concentration of released Ca2+ (Δ[CaT]) and the peak rate of Ca2+ release ((d/dt)Δ[CaT]) differed about 3-fold between the fibre types (slow-twitch: Δ[CaT], 127 ± 7 μM; (d/dt)Δ[CaT], 70 ± 6 μM ms−1; fast-twitch: Δ[CaT], 346 ± 6 μM; (d/dt)Δ[CaT], 212 ± 4 μM ms−1). In contrast, the half-duration of (d/dt)Δ[CaT] was very similar in the two fibre types (slow-twitch, 1.8 ± 0.1 ms; fast-twitch, 1.6 ± 0.0 ms). When fibres were stimulated with a 5-shock train at 67 Hz, the peaks of (d/dt)Δ[CaT] in response to the second and subsequent shocks were much smaller than that due to the first shock; the later peaks, expressed as a fraction of the amplitude of the first peak, were similar in the two fibre types (slow-twitch, 0.2–0.3; fast-twitch, 0.1–0.3). The results support the conclusion that individual SR Ca2+ release units function similarly in slow-twitch and fast-twitch mammalian fibres.
Konishi M. - One of the best experts on this subject based on the ideXlab platform.
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Basal intracellular free Mg2+ concentration in smooth muscle cells of guinea pig tenia cecum: intracellular calibration of the fluorescent indicator Furaptra
The Biophysical Society. Published by Elsevier Inc., 1997Co-Authors: Tashiro M., Konishi M.Abstract:Longitudinal muscle strips dissected from tenia cecum of guinea pig were loaded with the Mg2+ indicator, Furaptra, and the relation between the fluorescent ratio signal (R) and cytoplasmic free Mg2+ concentration ([Mg2+]i) was studied in smooth muscle cells at 25 degrees C. After the application of ionophores (4-bromo-A23187, monensin, and nigericin), a small immediate offset of R (deltaRjump) was followed by a slow change in R (deltaRslow), which reached a steady level within 2–5 h. The deltaRjump was independent of Mg2+ concentration in solution ([Mg2+]o), and was thought to be unrelated to the change in [Mg2+]i. The direction of the deltaRslow depended on [Mg2+]o with a reversal at approximately 1 mM [Mg2+]o. The intracellular calibration curve was constructed from the steady levels of deltaRslow, and the dissociation constant was 5.4 mM. With the intracellular calibration curve and correction for the deltaRjump, basal [Mg2+], was estimated to be 0.98 +/- 0.05 mM (mean +/- SE, n = 12). When the same calibration was applied to A7r5 cells and rat ventricular myocytes, estimates of basal [Mg2+]i of these cells were 0.74 +/- 0.02 mM (n = 33) and 1.13 +/- 0.06 mM (n = 9), respectively. These results suggest that the basal [Mg2+] level is approximately 1 mM at least in some types of smooth muscle cells, as generally found in striated muscles
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Na+ gradient-dependent Mg2+ transport in smooth muscle cells of guinea pig tenia cecum.
1997Co-Authors: Tashiro M., Konishi M.Abstract:Thin strips of guinea pig tenia cecum were loaded with the Mg2+ indicator Furaptra, and the indicator fluorescence signals measured in Ca2+-free condition were converted to cytoplasmic-free Mg2+ concentration ([Mg2+]i). Lowering the extracellular Na+ concentration ([Na+]o) caused a reversible increase in [Mg2+]i, consistent with the inhibition of Na+ gradient-dependent extrusion of cellular Mg2+ (Na+-Mg2+ exchange). Curve-fitting analysis indicated that the relation between [Na+]o and the rate of rise in [Mg2+], had a Hill coefficient of approximately 3, a [Na+]o at the half-maximal rate of rise of approximately 30 mM, and a maximal rate of 0.16 +/- 0.01 microM/s (mean +/- SE, n = 6). Depolarization with 56 mM K+ shifted the curve slightly toward higher [Na+]o without significantly changing the maximal rate, suggesting that the Na+-Mg2+ exchange was inhibited by depolarization. The maximal rate would correspond to a flux of 0.15-0.4 pmol/cm2/s, if cytoplasmic Mg2+ buffering power (defined as the ratio of the changes in total Mg2+ and free Mg2+ concentrations) is assumed to be 2-5. Ouabain (1-5 microM) increased the intracellular Na+ concentration, as assessed with fluorescence of SBFI (sodium-binding benzofuran isophthalate, a Na+ indicator), and elevated [Mg2+]i. In ouabain-treated preparations, removal of extracellular Na+ rapidly increased [Mg2+]i, with an initial rate of rise roughly proportional to the degree of the Mg2+ load, and, probably, to the Na+ load caused by ouabain. The enhanced rate of rise in [Mg2+]i (up to approximately 1 microM/s) could be attributed to the Mg2+ influx as a result of the reversed Na+-Mg2+ exchange. Our results support the presence of a reversible and possibly electrogenic Na+-Mg2+ exchange in the smooth muscle cells of tenia cecum
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Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators
The Biophysical Society. Published by Elsevier Inc., 1993Co-Authors: Konishi M.Abstract:Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or Furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or Furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in Furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and Furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and Furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and Furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50–100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and Furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters
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Ca transients in cardiac myocytes measured with a low affinity fluorescent indicator, Furaptra
The Biophysical Society. Published by Elsevier Inc., 1993Co-Authors: Konishi M.Abstract:Intracellular calcium ion ([Ca2+]i) transients were measured in single rat ventricular myocytes with the fluorescent indicator Furaptra. Cells were voltage clamped with a single patch electrode containing the K+ salt of Furaptra and fluorescence at 500 nm was measured during illumination with 350 and 370 nm light. Depolarizing voltage-clamp pulses elicited [Ca2+]-dependent fluorescent transients in 30 of 33 cells tested. The peak change in [Ca2+]i elicited by 50-ms depolarizations from -70 to +10 mV was 1.52 +/- 0.25 microM (mean +/- SEM, n = 7). The size of the [Ca2+]i transient increased in response to 10 microM isoproterenol, prolongation of the depolarization, and increasing pipette [Na+]. Because Furaptra is sensitive to Ca2+ and Mg2+, changes in [Mg2+]i during the [Ca2+]i transient could not be measured. Instead, a single-compartment model was developed to simulate changes in [Mg2+] during [Ca2+] transients. The simulations predicted that a 2 microM [Ca2+] transient was accompanied by a slow increase in [Mg2+] (14–29 microM), which became larger as basal [Mg2+] increased (0.5–2.0 mM). The [Mg2+] transient reached a peak approximately 1 s after the peak of the [Ca2+] transient with the slow changes in [Mg2+] dominated by competition at the Ca2+/Mg2+ sites of Troponin. These changes in [Mg2+], however, were so small and slow that they were unlikely to affect the Furaptra fluorescence signal at the peak of the [Ca2+]i transient. The [Ca2+]i transient reported by Furaptra appears to be larger than that reported by other Ca2+ indicators; however, we conclude this larger transient is at least as accurate as [Ca2+]i transients reported by the other indicators
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Fluorescence signals from the Mg2+/Ca2+ indicator Furaptra in frog skeletal muscle fibers
The Biophysical Society. Published by Elsevier Inc., 1993Co-Authors: Konishi M., Suda N., Kurihara S.Abstract:The fluorescent Mg2+/Ca2+ indicator, Furaptra, was injected into single frog skeletal muscle fibers, and the indicator's fluorescence signals were measured and analyzed with particular interest in the free Mg2+ concentration ([Mg2+]) in resting muscle. Based on the fluorescence excitation spectrum of Furaptra, the calibrated myoplasmic [Mg2+] level averaged 0.54 mM, if the value of dissociation constant (KD) for Mg2+ obtained in vitro (5.5 mM) was used. However, if the indicator reacts with Mg2+ with a two-fold larger KD in myoplasm, as previously suggested for the Furaptra-Ca2+ reaction (M. Konishi, S. Hollingworth, A.B. Harkins, S.M. Baylor. 1991. J. Gen. Physiol. 97:271–301), the calculated [Mg2+] would average 1.1 mM. Thus, the value 1.1 mM probably represents the best estimate from Furaptra of [Mg2+] in resting muscle fibers. Extracellular perfusion of muscle fibers with high Mg2+ concentration solution or low Na+ concentration solution did not cause any detectable changes in the [Mg2+]-related Furaptra fluorescence within 4min. The results suggest that the myoplasmic [Mg2+] is highly regulated near the resting level of 1 mM, and that changes only occur with a very slow time course
Konishi Masato - One of the best experts on this subject based on the ideXlab platform.
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Physiological Pathway of Magnesium Influx in Rat Ventricular Myocytes
Biophysical Society. Published by Elsevier Inc., 2014Co-Authors: Tashiro Michiko, Inoue Hana, Konishi MasatoAbstract:AbstractCytoplasmic free Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with a fluorescent indicator Furaptra (mag-fura-2) introduced by AM-loading. By incubation of the cells in a high-K+ (Ca2+- and Mg2+-free) solution, [Mg2+]i decreased from ∼ 0.9 mM to 0.2 to 0.5 mM. The lowered [Mg2+]i was recovered by perfusion with Ca2+-free Tyrode’s solution containing 1 mM Mg2+. The time course of the [Mg2+]i recovery was fitted by a single exponential function, and the first derivative at time 0 was analyzed as being proportional to the initial Mg2+ influx rate. The Mg2+ influx rate was inversely related to [Mg2+]i, being higher at low [Mg2+]i. The Mg2+ influx rate was augmented by the high extracellular Mg2+ concentration (5 mM), whereas it was greatly reduced by cell membrane depolarization caused by high K+. Known inhibitors of TRPM7 channels, 2-aminoethoxydiphenyl borate (2-APB), NS8593, and spermine reduced the Mg2+ influx rate with half inhibitory concentrations (IC50) of, respectively, 17 μM, 2.0 μM, and 22 μM. We also studied Ni2+ influx by fluorescence quenching of intracellular Furaptra by Ni2+. The Ni2+ influx was activated by lowering intra- and extracellular Mg2+ concentrations, and it was inhibited by 2-APB and NS8593 with IC50 values comparable with those for the Mg2+ influx. Intracellular alkalization (caused by pulse application of NH4Cl) enhanced, whereas intracellular acidification (induced after the removal of NH4Cl) slowed the Mg2+ influx. Under the whole-cell patch-clamp configuration, the removal of intracellular and extracellular divalent cations induced large inward and outward currents, MIC (Mg-inhibited cation) currents or IMIC, carried by monovalent cations likely via TRPM7 channels. IMIC measured at -120 mV was diminished to ∼ 50% by 100 μM 2-APB or 10 μM NS8593. These results suggest that TRPM7/MIC channels serve as a major physiological pathway of Mg2+ influx in rat ventricular myocytes
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Metabolic Inhibition Strongly Inhibits Na+-Dependent Mg2+ Efflux in Rat Ventricular Myocytes
Biophysical Society. Published by Elsevier Inc., 2009Co-Authors: Tashiro Michiko, Inoue Hana, Konishi MasatoAbstract:AbstractWe measured intracellular Mg2+ concentration ([Mg2+]i) in rat ventricular myocytes using the fluorescent indicator Furaptra (25°C). In normally energized cells loaded with Mg2+, the introduction of extracellular Na+ induced a rapid decrease in [Mg2+]i: the initial rate of decrease in [Mg2+]i (initial Δ[Mg2+]i/Δt) is thought to represent the rate of Na+-dependent Mg2+ efflux (putative Na+/Mg2+ exchange). To determine whether Mg2+ efflux depends directly on energy derived from cellular metabolism, in addition to the transmembrane Na+ gradient, we estimated the initial Δ[Mg2+]i/Δt after metabolic inhibition. In the absence of extracellular Na+ and Ca2+, treatment of the cells with 1 μM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, an uncoupler of mitochondria, caused a large increase in [Mg2+]i from ∼0.9 mM to ∼2.5 mM in a period of 5–8 min (probably because of breakdown of MgATP and release of Mg2+) and cell shortening to ∼50% of the initial length (probably because of formation of rigor cross-bridges). Similar increases in [Mg2+]i and cell shortening were observed after application of 5 mM potassium cyanide (KCN) (an inhibitor of respiration) for ≥90 min. The initial Δ[Mg2+]i/Δt was diminished, on average, by 90% in carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone-treated cells and 92% in KCN-treated cells. When the cells were treated with 5 mM KCN for shorter times (59–85 min), a significant decrease in the initial Δ[Mg2+]i/Δt (on average by 59%) was observed with only a slight shortening of the cell length. Intracellular Na+ concentration ([Na+]i) estimated with a Na+ indicator sodium-binding benzofuran isophthalate was, on average, 5.0–10.5 mM during the time required for the initial Δ[Mg2+]i/Δt measurements, which is well below the [Na+]i level for half inhibition of the Mg2+ efflux (∼40 mM). Normalization of intracellular pH using 10 μM nigericin, a H+ ionophore, did not reverse the inhibition of the Mg2+ efflux. From these results, it seems likely that a decrease in ATP below the threshold of rigor cross-bridge formation (∼0.4 mM estimated indirectly in the this study), rather than elevation of [Na+]i or intracellular acidosis, inhibits the Mg2+ efflux, suggesting the absolute necessity of ATP for the Na+/Mg2+ exchange
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Effects of Intracellular and Extracellular Concentrations of Ca2+, K+, and Cl− on the Na+-Dependent Mg2+ Efflux in Rat Ventricular Myocytes
The Biophysical Society. Published by Elsevier Inc., 2006Co-Authors: Tashiro Michiko, Tursun Pulat, Miyazaki Takefumi, Watanabe Masaru, Konishi MasatoAbstract:AbstractIntracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator Furaptra (25°C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Δ[Mg2+]i/Δt) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Δ[Mg2+]i/Δt values with 140mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1mM (107.3±8.7% of the control value measured at 0mM [Ca2+]o in the presence of 0.1mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5μM for 3.5h) did not significantly change the initial Δ[Mg2+]i/Δt: 115.2±7.5% (seven BAPTA-loaded cells) and 109.5±10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl− were modified under constant [Na+]o (70mM), [Ca2+]o (0mM with 0.1mM EGTA), and membrane potential (–13mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Δ[Mg2+]i/Δt: 1), changes in [K+]o between 0mM and 75mM (65.6±5.0% (n=11) and 79.0±6.0% (n=8), respectively, of the control values measured at 140mM [Na+]o without any modification of extracellular and intracellular K+ and Cl−); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7±8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl−-free solutions (71.6±5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl−, in cardiac myocytes
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Effects of Intracellular and Extracellular Concentrations of Ca(2+), K(+), and Cl(−) on the Na(+)-Dependent Mg(2+) Efflux in Rat Ventricular Myocytes
Biophysical Society, 2006Co-Authors: Tashiro Michiko, Tursun Pulat, Miyazaki Takefumi, Watanabe Masaru, Konishi MasatoAbstract:Intracellular Mg(2+) concentration ([Mg(2+)](i)) was measured in rat ventricular myocytes with the fluorescent indicator Furaptra (25°C). After the myocytes were loaded with Mg(2+), the initial rate of decrease in [Mg(2+)](i) (initial Δ[Mg(2+)](i)/Δt) was estimated upon introduction of extracellular Na(+), as an index of the rate of Na(+)-dependent Mg(2+) efflux. The initial Δ[Mg(2+)](i)/Δt values with 140 mM [Na(+)](o) were essentially unchanged by the addition of extracellular Ca(2+) up to 1 mM (107.3 ± 8.7% of the control value measured at 0 mM [Ca(2+)](o) in the presence of 0.1 mM EGTA, n = 5). Intracellular loading of a Ca(2+) chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 μM for 3.5 h) did not significantly change the initial Δ[Mg(2+)](i)/Δt: 115.2 ± 7.5% (seven BAPTA-loaded cells) and 109.5 ± 10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K(+) and Cl(−) were modified under constant [Na(+)](o) (70 mM), [Ca(2+)](o) (0 mM with 0.1 mM EGTA), and membrane potential (–13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Δ[Mg(2+)](i)/Δt: 1), changes in [K(+)](o) between 0 mM and 75 mM (65.6 ± 5.0% (n = 11) and 79.0 ± 6.0% (n = 8), respectively, of the control values measured at 140 mM [Na(+)](o) without any modification of extracellular and intracellular K(+) and Cl(−)); 2), intracellular perfusion with K(+)-free (Cs(+)-substituted) solution from the patch pipette in combination with removal of extracellular K(+) (77.7 ± 8.2%, n = 8); and 3), extracellular and intracellular perfusion with K(+)-free and Cl(−)-free solutions (71.6 ± 5.1%, n = 5). These results suggest that Mg(2+) is transported in exchange with Na(+), but not with Ca(2+), K(+), or Cl(−), in cardiac myocytes
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Intracellular and Extracellular Concentrations of Na+ Modulate Mg2+ Transport in Rat Ventricular Myocytes
The Biophysical Society. Published by Elsevier Inc., 2005Co-Authors: Tashiro Michiko, Tursun Pulat, Konishi MasatoAbstract:AbstractApparent free cytoplasmic concentrations of Mg2+ ([Mg2+]i) and Na+ ([Na+]i) were estimated in rat ventricular myocytes using fluorescent indicators, Furaptra (mag-fura-2) for Mg2+ and sodium-binding benzofuran isophthalate for Na+, at 25°C in Ca2+-free conditions. Analysis included corrections for the influence of Na+ on Furaptra fluorescence found in vitro and in vivo. The myocytes were loaded with Mg2+ in a solution containing 24mM Mg2+ either in the presence of 106mM Na+ plus 1mM ouabain (Na+ loading) or in the presence of only 1.6mM Na+ to deplete the cells of Na+ (Na+ depletion). The initial rate of decrease in [Mg2+]i from the Mg2+-loaded cells was estimated in the presence of 140mM Na+ and 1mM Mg2+ as an index of the rate of extracellular Na+-dependent Mg2+ efflux. Average [Na+]i, when estimated from sodium-binding benzofuran isophthalate fluorescence in separate experiments, increased from 12 to 31mM and 47mM after Na+ loading for 1 and 3h, respectively, and decreased to ∼0mM after 3h of Na+ depletion. The intracellular Na+ loading significantly reduced the initial rate of decrease in [Mg2+]i, on average, by 40% at 1h and by 64% at 3h, suggesting that the Mg2+ efflux was inhibited by intracellular Na+ with 50% inhibition at ∼40mM. A reduction of the rate of Mg2+ efflux was also observed when Na+ was introduced into the cells through the amphotericin B-perforated cell membrane (perforated patch-clamp technique) via a patch pipette that contained 130mM Na+. When the cells were heavily loaded with Na+ with ouabain in combination with intracellular perfusion from the patch pipette containing 130mM Na+, removal of extracellular Na+ caused an increase in [Mg2+]i, albeit at a very limited rate, which could be interpreted as reversal of the Mg2+ transport, i.e., Mg2+ influx driven by reversed Na+ gradient. Extracellular Na+ dependence of the rate of Mg2+ efflux revealed that the Mg2+ efflux was activated by extracellular Na+ with half-maximal activation at 55mM. These results contribute to a quantitative characterization of the Na+-Mg2+ exchange in cardiac myocytes
