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Frog alpha- and beta-ryanodine receptors provide distinct intracellular Ca2+ signals in a myogenic cell line.

Kashiyama T, Murayama T, Suzuki E, Allen PD, Ogawa Y - PLoS ONE (2010)

Bottom Line: Furthermore, procaine did not affect the peak height of high [K(+)](o)-induced Ca(2+) transients, suggesting minor amplification of Ca(2+) release by beta-RyR via CICR in 1B5 myotubes.These findings suggest that alpha-RyR and beta-RyR provide distinct intracellular Ca(2+) signals in a myogenic cell line.These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Juntendo University School of Medicine, Tokyo, Japan.

ABSTRACT

Background: In frog skeletal muscle, two ryanodine receptor (RyR) isoforms, alpha-RyR and beta-RyR, are expressed in nearly equal amounts. However, the roles and significance of the two isoforms in excitation-contraction (E-C) coupling remains to be elucidated.

Methodology/principal findings: In this study, we expressed either or both alpha-RyR and beta-RyR in 1B5 RyR-deficient myotubes using the herpes simplex virus 1 helper-free amplicon system. Immunological characterizations revealed that alpha-RyR and beta-RyR are appropriately expressed and targeted at the junctions in 1B5 myotubes. In Ca(2+) imaging studies, each isoform exhibited caffeine-induced Ca(2+) transients, an indicative of Ca(2+)-induced Ca(2+) release (CICR). However, the fashion of Ca(2+) release events was fundamentally different: alpha-RyR mediated graded and sustained Ca(2+) release observed uniformly throughout the cytoplasm, whereas beta-RyR supported all-or-none type regenerative Ca(2+) oscillations and waves. alpha-RyR but not beta-RyR exhibited Ca(2+) transients triggered by membrane depolarization with high [K(+)](o) that were nifedipine-sensitive, indicating that only alpha-RyR mediates depolarization-induced Ca(2+) release. Myotubes co-expressing alpha-RyR and beta-RyR demonstrated high [K(+)](o)-induced Ca(2+) transients which were indistinguishable from those with myotubes expressing alpha-RyR alone. Furthermore, procaine did not affect the peak height of high [K(+)](o)-induced Ca(2+) transients, suggesting minor amplification of Ca(2+) release by beta-RyR via CICR in 1B5 myotubes.

Conclusions/significance: These findings suggest that alpha-RyR and beta-RyR provide distinct intracellular Ca(2+) signals in a myogenic cell line. These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.

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Caffeine-induced Ca2+ release.A, B. Myotubes expressing α-RyR (A) or β-RyR (B) were stimulated by varied concentrations (0.2–20 mM) of caffeine. Traces of three representative cells are shown. Sustained Ca2+ transients with no or only slight decline were observed with myotubes expressing α-RyR, whereas Ca2+ oscillations with rapid rise and fall phases were seen with myotubes expressing β-RyR. C. Average maximal changes in fluo-4 fluorescence (ΔF/F0) of responding cells plotted against caffeine concentration (mean ± SE, n = 34 and 41 for α-RyR and β-RyR, respectively). Open circles, α-RyR; filled circles, β-RyR. In cells expressing α-RyR, Ca2+ transient amplitudes increased with increasing caffeine concentrations, whereas the responses in cells expressing β-RyR after reaching threshold were independent of caffeine concentration. D. % myotubes responding to each caffeine concentration. Open circles, α-RyR (n = 34); filled circles, β-RyR (n = 41). All the myotubes expressing α-RyR responded to caffeine with graded magnitude, whereas β-RyR myotubes showed all-or-none responses with increasing fractions of responding cells as the caffeine concentration increased.
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pone-0011526-g003: Caffeine-induced Ca2+ release.A, B. Myotubes expressing α-RyR (A) or β-RyR (B) were stimulated by varied concentrations (0.2–20 mM) of caffeine. Traces of three representative cells are shown. Sustained Ca2+ transients with no or only slight decline were observed with myotubes expressing α-RyR, whereas Ca2+ oscillations with rapid rise and fall phases were seen with myotubes expressing β-RyR. C. Average maximal changes in fluo-4 fluorescence (ΔF/F0) of responding cells plotted against caffeine concentration (mean ± SE, n = 34 and 41 for α-RyR and β-RyR, respectively). Open circles, α-RyR; filled circles, β-RyR. In cells expressing α-RyR, Ca2+ transient amplitudes increased with increasing caffeine concentrations, whereas the responses in cells expressing β-RyR after reaching threshold were independent of caffeine concentration. D. % myotubes responding to each caffeine concentration. Open circles, α-RyR (n = 34); filled circles, β-RyR (n = 41). All the myotubes expressing α-RyR responded to caffeine with graded magnitude, whereas β-RyR myotubes showed all-or-none responses with increasing fractions of responding cells as the caffeine concentration increased.

Mentions: Fig. 3 demonstrates caffeine-induced Ca2+ transients of myotubes expressing α-RyR or β-RyR. Myotubes expressing α-RyR exhibited dose-dependent Ca2+ transients in response to caffeine with only a slight decline in signal during a 30 sec exposure (Fig. 3A). The peak amplitude of the caffeine-induced Ca2+ release increased with caffeine dose and saturated around 10 mM (Fig. 3A, 3C). Myotubes expressing β-RyR exhibited Ca2+ oscillations with rapid rise and fall phases (like a burst of spikes) during exposure to caffeine (0.2–20 mM) (Fig. 3B). Notably, there are large cell-to-cell variations in threshold concentrations for caffeine. The individual Ca2+ transients tended to be longer in duration at a higher caffeine dose, and some cells exhibited long-lasting (>10 sec) Ca2+ transients when the caffeine concentration was 5 mM or higher. The peak amplitude of Ca2+ oscillations was constantly high in an all-or-none fashion, irrespective of caffeine dose (Fig. 3C). The peak amplitude of β-RyR is greater than the maximum response of α-RyR, which shows a sustained Ca2+ release (Fig. 3B, 3C). The fraction of responding cells increased with caffeine dose (Fig. 3D). This is in marked contrast to myotubes expressing α-RyR, where all expressing myotubes responded to the caffeine doses examined in a graded manner (Fig. 3A, 3D).


Frog alpha- and beta-ryanodine receptors provide distinct intracellular Ca2+ signals in a myogenic cell line.

Kashiyama T, Murayama T, Suzuki E, Allen PD, Ogawa Y - PLoS ONE (2010)

Caffeine-induced Ca2+ release.A, B. Myotubes expressing α-RyR (A) or β-RyR (B) were stimulated by varied concentrations (0.2–20 mM) of caffeine. Traces of three representative cells are shown. Sustained Ca2+ transients with no or only slight decline were observed with myotubes expressing α-RyR, whereas Ca2+ oscillations with rapid rise and fall phases were seen with myotubes expressing β-RyR. C. Average maximal changes in fluo-4 fluorescence (ΔF/F0) of responding cells plotted against caffeine concentration (mean ± SE, n = 34 and 41 for α-RyR and β-RyR, respectively). Open circles, α-RyR; filled circles, β-RyR. In cells expressing α-RyR, Ca2+ transient amplitudes increased with increasing caffeine concentrations, whereas the responses in cells expressing β-RyR after reaching threshold were independent of caffeine concentration. D. % myotubes responding to each caffeine concentration. Open circles, α-RyR (n = 34); filled circles, β-RyR (n = 41). All the myotubes expressing α-RyR responded to caffeine with graded magnitude, whereas β-RyR myotubes showed all-or-none responses with increasing fractions of responding cells as the caffeine concentration increased.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2902508&req=5

pone-0011526-g003: Caffeine-induced Ca2+ release.A, B. Myotubes expressing α-RyR (A) or β-RyR (B) were stimulated by varied concentrations (0.2–20 mM) of caffeine. Traces of three representative cells are shown. Sustained Ca2+ transients with no or only slight decline were observed with myotubes expressing α-RyR, whereas Ca2+ oscillations with rapid rise and fall phases were seen with myotubes expressing β-RyR. C. Average maximal changes in fluo-4 fluorescence (ΔF/F0) of responding cells plotted against caffeine concentration (mean ± SE, n = 34 and 41 for α-RyR and β-RyR, respectively). Open circles, α-RyR; filled circles, β-RyR. In cells expressing α-RyR, Ca2+ transient amplitudes increased with increasing caffeine concentrations, whereas the responses in cells expressing β-RyR after reaching threshold were independent of caffeine concentration. D. % myotubes responding to each caffeine concentration. Open circles, α-RyR (n = 34); filled circles, β-RyR (n = 41). All the myotubes expressing α-RyR responded to caffeine with graded magnitude, whereas β-RyR myotubes showed all-or-none responses with increasing fractions of responding cells as the caffeine concentration increased.
Mentions: Fig. 3 demonstrates caffeine-induced Ca2+ transients of myotubes expressing α-RyR or β-RyR. Myotubes expressing α-RyR exhibited dose-dependent Ca2+ transients in response to caffeine with only a slight decline in signal during a 30 sec exposure (Fig. 3A). The peak amplitude of the caffeine-induced Ca2+ release increased with caffeine dose and saturated around 10 mM (Fig. 3A, 3C). Myotubes expressing β-RyR exhibited Ca2+ oscillations with rapid rise and fall phases (like a burst of spikes) during exposure to caffeine (0.2–20 mM) (Fig. 3B). Notably, there are large cell-to-cell variations in threshold concentrations for caffeine. The individual Ca2+ transients tended to be longer in duration at a higher caffeine dose, and some cells exhibited long-lasting (>10 sec) Ca2+ transients when the caffeine concentration was 5 mM or higher. The peak amplitude of Ca2+ oscillations was constantly high in an all-or-none fashion, irrespective of caffeine dose (Fig. 3C). The peak amplitude of β-RyR is greater than the maximum response of α-RyR, which shows a sustained Ca2+ release (Fig. 3B, 3C). The fraction of responding cells increased with caffeine dose (Fig. 3D). This is in marked contrast to myotubes expressing α-RyR, where all expressing myotubes responded to the caffeine doses examined in a graded manner (Fig. 3A, 3D).

Bottom Line: Furthermore, procaine did not affect the peak height of high [K(+)](o)-induced Ca(2+) transients, suggesting minor amplification of Ca(2+) release by beta-RyR via CICR in 1B5 myotubes.These findings suggest that alpha-RyR and beta-RyR provide distinct intracellular Ca(2+) signals in a myogenic cell line.These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Juntendo University School of Medicine, Tokyo, Japan.

ABSTRACT

Background: In frog skeletal muscle, two ryanodine receptor (RyR) isoforms, alpha-RyR and beta-RyR, are expressed in nearly equal amounts. However, the roles and significance of the two isoforms in excitation-contraction (E-C) coupling remains to be elucidated.

Methodology/principal findings: In this study, we expressed either or both alpha-RyR and beta-RyR in 1B5 RyR-deficient myotubes using the herpes simplex virus 1 helper-free amplicon system. Immunological characterizations revealed that alpha-RyR and beta-RyR are appropriately expressed and targeted at the junctions in 1B5 myotubes. In Ca(2+) imaging studies, each isoform exhibited caffeine-induced Ca(2+) transients, an indicative of Ca(2+)-induced Ca(2+) release (CICR). However, the fashion of Ca(2+) release events was fundamentally different: alpha-RyR mediated graded and sustained Ca(2+) release observed uniformly throughout the cytoplasm, whereas beta-RyR supported all-or-none type regenerative Ca(2+) oscillations and waves. alpha-RyR but not beta-RyR exhibited Ca(2+) transients triggered by membrane depolarization with high [K(+)](o) that were nifedipine-sensitive, indicating that only alpha-RyR mediates depolarization-induced Ca(2+) release. Myotubes co-expressing alpha-RyR and beta-RyR demonstrated high [K(+)](o)-induced Ca(2+) transients which were indistinguishable from those with myotubes expressing alpha-RyR alone. Furthermore, procaine did not affect the peak height of high [K(+)](o)-induced Ca(2+) transients, suggesting minor amplification of Ca(2+) release by beta-RyR via CICR in 1B5 myotubes.

Conclusions/significance: These findings suggest that alpha-RyR and beta-RyR provide distinct intracellular Ca(2+) signals in a myogenic cell line. These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.

Show MeSH
Related in: MedlinePlus