scholarly journals Excitation of skinned muscle fibers by imposed ion gradients. I. Stimulation of 45Ca efflux at constant [K][Cl] product.

1985 ◽  
Vol 86 (6) ◽  
pp. 813-832 ◽  
Author(s):  
E W Stephenson
Keyword(s):  
Isometric Force ◽  
Muscle Fibers ◽  
Water Flux ◽  
Skinned Muscle ◽  
Skinned Muscle Fibers ◽  
Diffusion Potentials ◽  
45Ca Efflux ◽  
Osmotic Effects ◽  
45Ca Release ◽  
And Diffusion

45Ca efflux from skinned muscle fibers is stimulated transiently, by a highly Ca2+-dependent mechanism, by KCl replacement of K propionate. In the present studies, Cl replaced the much less permeant anion methanesulfonate (Mes) either (a) at constant [K], in which increased [K][Cl] permits net KCl and water flux across internal membranes, or (b) at constant [K][Cl] (choline substitution), in which the imposed gradients and diffusion potentials should dissipate slowly. 45Ca efflux and isometric force were measured simultaneously on segments of frog semitendinosus fibers skinned by microdissection. EGTA was applied to chelate released 45Ca either (a) shortly after high [Cl] (interrupted response), to minimize reaccumulation, (b) before high [Cl] (pretreated response), to evaluate Ca2+ dependence, or (c) under control conditions in KMes. KCl replacement of KMes stimulated release of 65% fiber 45Ca within 1 min in interrupted responses; EGTA pretreatment was only moderately inhibitory with substantial residual stimulation. In contrast, choline Cl replacement of KMes induced release of 26-35% fiber 45Ca in interrupted responses; EGTA pretreatment was strongly inhibitory, but release significantly exceeded control with a small, sustained increase in Ca2+-insensitive efflux. These differences in 45Ca release and EGTA inhibition suggest that Cl replacement of Mes at constant [K] stimulates efflux by osmotic effects as well as imposed diffusion potentials; at least half the stimulated 45Ca loss (above control) in interrupted KCl responses is attributable to an osmotic component with low Ca2+ sensitivity. In the highly Ca2+-sensitive stimulation at constant [K][Cl], 45Ca release (above control) in interrupted responses correlated well with that in the pretreated responses of segments from the same fiber, with a slope of 8.4. This relationship suggests that imposed diffusion potentials stimulate a small Ca2+-insensitive component that gradates a much larger Ca2+-dependent efflux. The Ca2+-insensitive component apparently reflects intermediate steps in the excitation-contraction coupling that require positive feedback to result in sufficient Ca release for contraction.

scholarly journals Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers.

1981 ◽  
Vol 77 (4) ◽  
pp. 419-443 ◽  
Author(s):  
E W Stephenson
Keyword(s):  
Isometric Force ◽  
Muscle Fibers ◽  
Frog Skeletal Muscle ◽  
Receptor Affinity ◽  
Skinned Muscle ◽  
Skinned Muscle Fibers ◽  
45Ca Efflux ◽  
45Ca Release ◽  
Stimulation Of

Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.

scholarly journals Properties of chloride-stimulated 45Ca flux in skinned muscle fibers.

1978 ◽  
Vol 71 (4) ◽  
pp. 411-430 ◽  
Author(s):  
E W Stephenson
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Time Course ◽  
Isometric Force ◽  
Frog Muscle ◽  
Published Data ◽  
Skinned Muscle ◽  
Skinned Muscle Fibers ◽  
45Ca Efflux ◽  
45Ca Release ◽  
Stimulation Of

Isometric force and 45Ca loss from fiber to bath were measured simultaneously in skinned fibers from frog muscle at 19 degrees C. In unstimulated fibers, 45Ca efflux from the sarcoplasmic reticulum (SR) was very slow, with little or no dependence on EGTA (0.1-5 mM) or Mg++ (20 micrometer-1.3 mM). Stimulation by high [Cl] at 0.11 mM Mg++ caused rapid force transients (duration approximately 10 s) and 45Ca release. This response was followed for 55 s, with 5 mM EGTA added to chelate myofilament space (MFS) Ca either (a) after relaxation, (b) near the peak of the force spike, or (c) before or with the stimulus. When EGTA was present during Cl application, stimulation of 45Ca release was undetectable. Analysis of the time-course of tracer loss during the three protocols showed that when EGTA was absent, 16% of the fiber tracer was released from the SR within approximately 3 s, and 70% of the tracer still in the MFS near the peak of the force spike was subsequently reaccumulated. The results suggest that (a) the Cl response is highly Ca-dependent; (b) stimulation increases 45Ca efflux from the SR at least 100-200-fold; and (c) the rate of reaccumulation is much slower than the influx predicted from published data on resting fibers, raising the possibility that depolarization inhibits active Ca transport by the SR.

scholarly journals Excitation of skinned muscle fibers by imposed ion gradients. II. Influence of quercetin and ATP removal on the Ca2+-insensitive component of stimulated 45Ca efflux.

1985 ◽  
Vol 86 (6) ◽  
pp. 833-852 ◽  
Author(s):  
E W Stephenson
Keyword(s):  
Adenine Nucleotide ◽  
Isometric Force ◽  
Muscle Fibers ◽  
Semitendinosus Muscle ◽  
Skinned Muscle ◽  
Skinned Muscle Fibers ◽  
Unstimulated Control ◽  
Dependent Component ◽  
Control Loss ◽  
45Ca Efflux

Ionic gradients imposed by choline Cl replacement of K methanesulfonate (Mes) at constant [K][Cl] product stimulate 45Ca efflux from skinned muscle fibers; a small, sustained Ca2+-insensitive efflux component, observed in EGTA, appears to grade a much larger Ca2+-dependent component responsible for contractile activation and is likely to reflect intermediate steps in excitation-contraction coupling. The present studies examined ATP-related effects on the Ca2+-insensitive stimulation. 45Ca efflux was measured on segments of frog semitendinosus muscle skinned by microdissection, with isometric force monitored continuously. The Ca2+-insensitive component was potentiated by quercetin, a flavonoid thought to inhibit the sarcoplasmic reticulum (SR) Ca pump by stabilizing a phosphorylated intermediate. Quercetin increased the stimulated net 45Ca release in the absence of EGTA, as expected from inhibition of reaccumulation, but its effectiveness in EGTA indicated potentiation of unidirectional efflux as such. Quercetin also increased unstimulated (control) 45Ca efflux in EGTA, to a smaller extent; potentiation appeared to be a function of efflux, with stimulation above control loss increased approximately 2.6-fold. ATP removal before stimulation, which led to rigor force and increased stiffness, prevented all quercetin effects in EGTA. ATP removal by itself inhibited ionic stimulation of the Ca2+-insensitive component, with little residual increase above the parallel control loss. Addition of the nonhydrolyzable ATP analogue AMP-PCP ([adenylyl-beta,gamma-methylene]diphosphate) (0.8 mM) after ATP removal gave similar results to ATP-free solution, which suggests that adenine nucleotide binding alone does not support stimulation by choline Cl. These results imply a fundamental role for ATP in the excitation of skinned fibers by imposed diffusion potentials; they also suggest that ATP regulates the SR Ca efflux channel, in a manner that could provide the positive feedback in Ca2+-dependent Ca release.

scholarly journals Influence of magnesium on chloride-induced calcium release in skinned muscle fibers.

1977 ◽  
Vol 69 (1) ◽  
pp. 17-35 ◽  
Author(s):  
E W Stephenson ◽  
R J Podolsky
Keyword(s):  
Calcium Release ◽  
Isometric Force ◽  
Muscle Fibers ◽  
Ringer Solution ◽  
Skinned Fibers ◽  
Skinned Muscle ◽  
Skinned Muscle Fibers ◽  
Ca Uptake ◽  
45Ca Release ◽  
Net Release

Chloride-induced Ca release in skinned muscle fibers was studied by measuring isometric force transients and 45Ca loss from fiber to washout solutions. Skinned fibers prepared from muscles soaked in normal Ringer solution made large force transients in 120 mM Cl solution with 5 mM ATP and 1 mM Mg, but 3 mM Mg was inhibitory. Mg inhibition was antagonized by low temperature and by Cd, agents which slow active Ca uptake by the sarcoplasmic reticulum (SR). In low Mg++, Cl stimulated rapid 45Ca release from the SR in sufficient amounts to account for the force response. The increased 45Ca release was inhibited by EGTA, suggesting that release requires free Ca under these conditions. The 45Ca initially released was partially reaccumulated later. Reaccumulation was increased in higher Mg++. These results provide additional evidence that the Ca uptake rate is an important determinant of net release, and suggest that Mg++ acts primarily on this mechanism. Skinned fibers prepared from muscles soaked in low Cl solutions could give force responses to Cl solutions with 3 mM and 6 mM Mg. This observation suggests that the Cl stimulus varies with the [Cl] gradient across the internal membranes, and supports the hypothesis that applied Cl causes membrane depolarization.

scholarly journals An Automated Apparatus for Isometric Force Analysis of Skinned Muscle Fibers

2009 ◽  
Vol 96 (3) ◽  
pp. 616a
Author(s):  
David Marquez ◽  
Matthew Tomlinson ◽  
Jeff Lievers ◽  
Jim Hartman ◽  
Alan Russell ◽  
...  
Keyword(s):  
Isometric Force ◽  
Muscle Fibers ◽  
Force Analysis ◽  
Skinned Muscle ◽  
Skinned Muscle Fibers

The role of free calcium ion in calcium release in skinned muscle fibers

1982 ◽  
Vol 60 (4) ◽  
pp. 417-426 ◽  
Author(s):  
Elizabeth W. Stephenson
Keyword(s):  
Calcium Release ◽  
Isometric Force ◽  
Signal Transmission ◽  
Muscle Fibers ◽  
Free Calcium ◽  
Control Analysis ◽  
Skinned Muscle ◽  
Skinned Muscle Fibers ◽  
Intracellular Ca ◽  
Active Transport System

Major questions in excitation–contraction coupling of fast skeletal muscle concern the mechanism of signal transmission between sarcolemma and sarcoplasmic reticulum (SR), the mechanism of SR Ca release, and operation of the SR active transport system during excitation. Intracellular Ca movement can be studied in skinned muscle fibers with more direct control, analysis of 45Ca flux, and simultaneous isometric force measurements. Ca release can be stimulated by bath Ca2+ itself, ionic "depolarization," Mg2+ reduction, or caffeine. The effectiveness of bath Ca2+ has suggested a possible role for Ca2+ in physiological release, but this response is difficult to analyze and evaluate. Related evidence emerged from analysis of other responses: with all agents studied, stimulation of 45Ca efflux is highly Ca2+-dependent. The presence of a Ca chelator prevents detectable stimulation by ionic "depolarization" or Mg2+ reduction and inhibits the potent caffeine stimulus; inhibition is graded with chelator concentration and caffeine concentration, and is synergistic with inhibition by increased Mg2+. The results indicate that a Ca2+-dependent pathway mediates most or all of stimulated 45Ca efflux in skinned fibers, and has properties compatible with a function in physiological Ca release.

scholarly journals Regulation by magnesium of intracellular calcium movement in skinned muscle fibers.

1977 ◽  
Vol 69 (1) ◽  
pp. 1-16 ◽  
Author(s):  
E W Stephenson ◽  
R J Podolsky
Keyword(s):  
Isometric Force ◽  
Muscle Fibers ◽  
Force Development ◽  
Skinned Muscle ◽  
Skinned Muscle Fibers ◽  
Large Force ◽  
Ca Uptake ◽  
Transient Relaxation ◽  
Steady Force

The effect of Mg on Ca movement between the sarcoplasmic reticulum (SR) and myofilament space (MFS) was studied in skinned muscle fibers by using isometric force as an indicator of MFS Ca. In Ca-loaded fibers at 20 degrees C, the large force spike induced by Ca in 1 mM Mg (5 mM ATP) was strongly inhibited in 3 mM Mg, and force development was extremely slow. After a brief Ca stimulus in 1 mM Mg, relaxation in Ca-free solution was significantly faster in 3 mM Mg. These changes were due to altered Ca movements, since the effect of 3 mM Mg on steady force in CaEGTA solutions was small. Changes in Mg alone induced force transients apparently due to altered Ca movement. In relaxed fibers, decreasing the Mg to 0.25 mM caused phasic force development. In contracting fibers in Ca solutions, increasing the Mg caused a large transient relaxation. The effects of increased Mg were antagonized by 0.5 mM Cd, an inhibitor of the SR Ca transport system. The results indicate that active Ca uptake by the SR in situ is stimulated by Mg, and that it can affect local MFS [Ca++] in the presence of a substantial Ca source. These results provide evidence that an increased rate of Ca uptake in 3 mM Mg could account for inhibition of the large force spike associated with Ca-induced Ca release in skinned fibers.

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