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How source content determines intracellular Ca2+ release kinetics. Simultaneous measurement of [Ca2+] transients and [H+] displacement in skeletal muscle.

Pizarro G, Ríos E - J. Gen. Physiol. (2004)

Bottom Line: Steady release permeability (P), reached at the end of a 120-ms pulse, increased as Ca(SR) was progressively reduced by a prior conditioning pulse, reaching 2.34-fold at 25% of resting Ca(SR) (four cells).Peak P, reached early during a pulse, increased proportionally much less with SR depletion, decreasing at very low Ca(SR).These results are consistent with a major inhibitory effect of cytosolic (rather than intra-SR) Ca(2+) on the activity of Ca(2+) release channels.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Molecular Biophysics and Physiology, Rush University School of Medicine, 1750 W. Harrison St., Suite 1279JS, Chicago, IL 60612, USA.

ABSTRACT
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 to be strongly dependent on the Ca(2+) content in the sarcoplasmic reticulum (SR). In skeletal muscle, no such role is established. Seeking a robust measurement of Ca(2+) release and a way to reliably modify the SR content, we combined in the same cells the "EGTA/phenol red" method (Pape et al., 1995) to evaluate Ca(2+) release, with the "removal" method (Melzer et al., 1987) to evaluate release flux. The cytosol of voltage-clamped frog fibers was equilibrated with EGTA (36 mM), antipyrylazo III, and phenol red, and absorbance changes were monitored simultaneously at three wavelengths, affording largely independent evaluations of Delta[H(+)] and Delta[Ca(2+)] from which the amount of released Ca(2+) and the release flux were independently derived. Both methods yielded mutually consistent evaluations of flux. While the removal method gave a better kinetic picture of the release waveform, EGTA/phenol red provided continuous reproducible measures of calcium in the SR (Ca(SR)). Steady release permeability (P), reached at the end of a 120-ms pulse, increased as Ca(SR) was progressively reduced by a prior conditioning pulse, reaching 2.34-fold at 25% of resting Ca(SR) (four cells). Peak P, reached early during a pulse, increased proportionally much less with SR depletion, decreasing at very low Ca(SR). The increase in steady P upon depletion was associated with a slowing of the rate of decay of P after the peak (i.e., a slower inactivation of Ca(2+) release). These results are consistent with a major inhibitory effect of cytosolic (rather than intra-SR) Ca(2+) on the activity of Ca(2+) release channels.

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Initial analysis of optical records. (A) Changes in transmitted light intensity. I, simultaneously measured at three wavelengths as indicated. (B) Dye-related changes in absorbance at 575 and 720 nm, derived from intensities in A. Record ΔAAp(720) is derived (according to Eq. 4) by linear combination of ΔA(720) and ΔAintrinsic(720), which in turn is derived from ΔI(550), blue in A, and I(550). ΔAPR(575) (dashed) is derived according to Eq. 6, by linear combination of A(575), AAp(575), and Aintrinsic(575) (in turn evaluated by Eq. 5). AAp(575) is calculated as the sum of a resting component (second term, right hand side of Eq. 2) and a change ΔAAp(575), derived by scaling ΔAAp(720) by 0.25. The record in red, continuous trace, is ΔA(575) after correction for the intrinsic absorbance change (i.e., the total change in absorbance due to both dyes). It is very similar to ΔAPR(575), which stresses that the interference between the two dyes is almost negligible. Identifier 1723, vertical diameter = 73 μm, [phenol red]= 2.20 mM, [ApIII]= 0.86 mM, pH 6.35.
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fig5: Initial analysis of optical records. (A) Changes in transmitted light intensity. I, simultaneously measured at three wavelengths as indicated. (B) Dye-related changes in absorbance at 575 and 720 nm, derived from intensities in A. Record ΔAAp(720) is derived (according to Eq. 4) by linear combination of ΔA(720) and ΔAintrinsic(720), which in turn is derived from ΔI(550), blue in A, and I(550). ΔAPR(575) (dashed) is derived according to Eq. 6, by linear combination of A(575), AAp(575), and Aintrinsic(575) (in turn evaluated by Eq. 5). AAp(575) is calculated as the sum of a resting component (second term, right hand side of Eq. 2) and a change ΔAAp(575), derived by scaling ΔAAp(720) by 0.25. The record in red, continuous trace, is ΔA(575) after correction for the intrinsic absorbance change (i.e., the total change in absorbance due to both dyes). It is very similar to ΔAPR(575), which stresses that the interference between the two dyes is almost negligible. Identifier 1723, vertical diameter = 73 μm, [phenol red]= 2.20 mM, [ApIII]= 0.86 mM, pH 6.35.

Mentions: [H+](t) and Δ[Ca2+](t) were derived respectively from the evolution of [H phenol red] and [Ca ApIII2], the corresponding equilibrium equations, and the conservation equations expressing total [ApIII] and [phenol red] as sums of concentrations of its ion-bound and ion-free forms. In turn, [H phenol red](t) and [Ca ApIII2](t) were derived from simultaneously recorded absorbance changes at three wavelengths, using Eq. 1 with parameters adjusted for the different wavelengths. This results in three equations, which together with the conservation equations of ApIII and phenol red allow for uniquely determining five functions (time courses of bound and free forms of each dye plus the intrinsic absorbance). While this is possible in principle with any three wavelengths, the use of 575, 720, and 850 nm minimizes interference and error. The basic protocol was to record light signals (changes in light intensity) upon voltage stimulation at these wavelengths. Levels of steady intensity were recorded at the same wavelengths before application of the stimulus. Time course of total intensity was reconstructed by sum of change plus steady level. Intensities were then converted to absorbance changes and linearly combined to derive pure signals from the two dyes. This is illustrated in Fig. 4 for the fiber whose dye concentrations are plotted in Fig. 3. Fig. 4 A plots intensity signals and B absorbance changes.


How source content determines intracellular Ca2+ release kinetics. Simultaneous measurement of [Ca2+] transients and [H+] displacement in skeletal muscle.

Pizarro G, Ríos E - J. Gen. Physiol. (2004)

Initial analysis of optical records. (A) Changes in transmitted light intensity. I, simultaneously measured at three wavelengths as indicated. (B) Dye-related changes in absorbance at 575 and 720 nm, derived from intensities in A. Record ΔAAp(720) is derived (according to Eq. 4) by linear combination of ΔA(720) and ΔAintrinsic(720), which in turn is derived from ΔI(550), blue in A, and I(550). ΔAPR(575) (dashed) is derived according to Eq. 6, by linear combination of A(575), AAp(575), and Aintrinsic(575) (in turn evaluated by Eq. 5). AAp(575) is calculated as the sum of a resting component (second term, right hand side of Eq. 2) and a change ΔAAp(575), derived by scaling ΔAAp(720) by 0.25. The record in red, continuous trace, is ΔA(575) after correction for the intrinsic absorbance change (i.e., the total change in absorbance due to both dyes). It is very similar to ΔAPR(575), which stresses that the interference between the two dyes is almost negligible. Identifier 1723, vertical diameter = 73 μm, [phenol red]= 2.20 mM, [ApIII]= 0.86 mM, pH 6.35.
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Related In: Results  -  Collection

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fig5: Initial analysis of optical records. (A) Changes in transmitted light intensity. I, simultaneously measured at three wavelengths as indicated. (B) Dye-related changes in absorbance at 575 and 720 nm, derived from intensities in A. Record ΔAAp(720) is derived (according to Eq. 4) by linear combination of ΔA(720) and ΔAintrinsic(720), which in turn is derived from ΔI(550), blue in A, and I(550). ΔAPR(575) (dashed) is derived according to Eq. 6, by linear combination of A(575), AAp(575), and Aintrinsic(575) (in turn evaluated by Eq. 5). AAp(575) is calculated as the sum of a resting component (second term, right hand side of Eq. 2) and a change ΔAAp(575), derived by scaling ΔAAp(720) by 0.25. The record in red, continuous trace, is ΔA(575) after correction for the intrinsic absorbance change (i.e., the total change in absorbance due to both dyes). It is very similar to ΔAPR(575), which stresses that the interference between the two dyes is almost negligible. Identifier 1723, vertical diameter = 73 μm, [phenol red]= 2.20 mM, [ApIII]= 0.86 mM, pH 6.35.
Mentions: [H+](t) and Δ[Ca2+](t) were derived respectively from the evolution of [H phenol red] and [Ca ApIII2], the corresponding equilibrium equations, and the conservation equations expressing total [ApIII] and [phenol red] as sums of concentrations of its ion-bound and ion-free forms. In turn, [H phenol red](t) and [Ca ApIII2](t) were derived from simultaneously recorded absorbance changes at three wavelengths, using Eq. 1 with parameters adjusted for the different wavelengths. This results in three equations, which together with the conservation equations of ApIII and phenol red allow for uniquely determining five functions (time courses of bound and free forms of each dye plus the intrinsic absorbance). While this is possible in principle with any three wavelengths, the use of 575, 720, and 850 nm minimizes interference and error. The basic protocol was to record light signals (changes in light intensity) upon voltage stimulation at these wavelengths. Levels of steady intensity were recorded at the same wavelengths before application of the stimulus. Time course of total intensity was reconstructed by sum of change plus steady level. Intensities were then converted to absorbance changes and linearly combined to derive pure signals from the two dyes. This is illustrated in Fig. 4 for the fiber whose dye concentrations are plotted in Fig. 3. Fig. 4 A plots intensity signals and B absorbance changes.

Bottom Line: Steady release permeability (P), reached at the end of a 120-ms pulse, increased as Ca(SR) was progressively reduced by a prior conditioning pulse, reaching 2.34-fold at 25% of resting Ca(SR) (four cells).Peak P, reached early during a pulse, increased proportionally much less with SR depletion, decreasing at very low Ca(SR).These results are consistent with a major inhibitory effect of cytosolic (rather than intra-SR) Ca(2+) on the activity of Ca(2+) release channels.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Molecular Biophysics and Physiology, Rush University School of Medicine, 1750 W. Harrison St., Suite 1279JS, Chicago, IL 60612, USA.

ABSTRACT
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 to be strongly dependent on the Ca(2+) content in the sarcoplasmic reticulum (SR). In skeletal muscle, no such role is established. Seeking a robust measurement of Ca(2+) release and a way to reliably modify the SR content, we combined in the same cells the "EGTA/phenol red" method (Pape et al., 1995) to evaluate Ca(2+) release, with the "removal" method (Melzer et al., 1987) to evaluate release flux. The cytosol of voltage-clamped frog fibers was equilibrated with EGTA (36 mM), antipyrylazo III, and phenol red, and absorbance changes were monitored simultaneously at three wavelengths, affording largely independent evaluations of Delta[H(+)] and Delta[Ca(2+)] from which the amount of released Ca(2+) and the release flux were independently derived. Both methods yielded mutually consistent evaluations of flux. While the removal method gave a better kinetic picture of the release waveform, EGTA/phenol red provided continuous reproducible measures of calcium in the SR (Ca(SR)). Steady release permeability (P), reached at the end of a 120-ms pulse, increased as Ca(SR) was progressively reduced by a prior conditioning pulse, reaching 2.34-fold at 25% of resting Ca(SR) (four cells). Peak P, reached early during a pulse, increased proportionally much less with SR depletion, decreasing at very low Ca(SR). The increase in steady P upon depletion was associated with a slowing of the rate of decay of P after the peak (i.e., a slower inactivation of Ca(2+) release). These results are consistent with a major inhibitory effect of cytosolic (rather than intra-SR) Ca(2+) on the activity of Ca(2+) release channels.

Show MeSH
Related in: MedlinePlus