Fall in intracellular Po 2 at the onset of contractions in Xenopus single skeletal muscle fibers

2001 ◽  
Vol 90 (5) ◽  
pp. 1871-1876 ◽  
Author(s):  
Michael C. Hogan

It remains uncertain whether the delayed onset of mitochondrial respiration on initiation of muscle contractions is related to O2 availability. The purpose of this research was to measure the kinetics of the fall in intracellular Po 2 at the onset of a contractile work period in rested and previously worked single skeletal muscle fibers. Intact single skeletal muscle fibers ( n = 11) from Xenopus laevis were dissected from the lumbrical muscle, injected with an O2-sensitive probe, mounted in a glass chamber, and perfused with Ringer solution (Po 2 = 32 ± 4 Torr and pH = 7.0) at 20°C. Intracellular Po 2 was measured in each fiber during a protocol consisting sequentially of 1-min rest; 3 min of tetanic contractions (1 contraction/2 s); 5-min rest; and, finally, a second 3-min contractile period identical to the first. Maximal force development and the fall in force (to 83 ± 2 vs. 86 ± 3% of maximal force development) in contractile periods 1 and 2, respectively, were not significantly different. The time delay (time before intracellular Po 2 began to decrease after the onset of contractions) was significantly greater ( P < 0.01) in the first contractile period (13 ± 3 s) compared with the second (5 ± 2 s), as was the time to reach 50% of the contractile steady-state intracellular Po 2(28 ± 5 vs. 18 ± 4 s, respectively). In Xenopus single skeletal muscle fibers, 1) the lengthy response time for the fall in intracellular Po 2 at the onset of contractions suggests that intracellular factors other than O2 availability determine the on-kinetics of oxidative phosphorylation and 2) a prior contractile period results in more rapid on-kinetics.

1999 ◽  
Vol 86 (2) ◽  
pp. 720-724 ◽  
Author(s):  
Michael C. Hogan

Values of skeletal muscle intracellular[Formula: see text] during conditions ranging from rest to maximal metabolic rates have been difficult to quantify. A method for measurement of intracellular[Formula: see text] in isolated single skeletal muscle fibers by using O2-dependent quenching of a phosphorescent-O2probe is described. Intact single skeletal muscle fibers from Xenopus laevis were dissected from the lumbrical muscle and mounted in a glass chamber containing Ringer solution at 20°C. The chamber was placed on the stage of an inverted microscope configured for epi-illumination. A solution containing palladium- meso-tetra (4-carboxyphenyl) porphine bound to bovine serum albumin was injected into single fibers by micropipette pressure injection. Phosphorescence-decay curves (average of 10 rapid flashes) were recorded every 7 s from single cells ( n = 24) in which respiration had been eliminated with NaCN, while the [Formula: see text]of the Ringer solution surrounding the cell was varied from 0 to 159 Torr. For each measurement, the phosphorescence lifetime was calculated at the varied extracellular [Formula: see text] by obtaining a best-fit estimate by using a monoexponential function. The phosphorescence lifetime varied from 40 to 70 μs at an extracellular[Formula: see text] of 159 Torr to 650–700 μs at 0 Torr. The phosphorescent lifetimes for the varied[Formula: see text] were used to calculate, by using the Stern-Volmer relationship, the phosphorescence-quenching constant (100 Torr−1 ⋅ s−1), and the phosphorescence lifetime in a zero-O2 environment (690 μs) for the phosphor within the intracellular environment. This technique demonstrates a novel method for determining intracellular[Formula: see text] in isolated single skeletal muscle fibers.


2005 ◽  
Vol 99 (1) ◽  
pp. 308-312 ◽  
Author(s):  
C. M. Stary ◽  
M. C. Hogan

The purpose of the present study was to test the hypothesis that a preceding contractile period in isolated single skeletal muscle fibers would attenuate the decrease in pH during an identical, subsequent contractile period, thereby reducing the rate of fatigue. Intact single skeletal muscle fibers ( n = 9) were isolated from Xenopus lumbrical muscle and incubated with the fluorescent cytosolic H+ indicator 2′,7′-bis-(2-carboxyethyl)-5( 6 )-carboxyfluorescein (BCECF) AM for 30 min. Two identical contractile periods were performed in each fiber, separated by a 1-h recovery period. Force and intracellular pH (pHi) fluorescence were measured simultaneously while fibers were stimulated (tetanic contractions of 350-ms trains with 70-Hz stimuli at 9 V) at progressively increasing frequencies (0.25, 0.33, 0.5, and 1 contraction/s) until the development of fatigue (to 60% initial force). No significant difference ( P < 0.05) was observed between the first and second contractile periods in initial force development, resting pHi, or time to fatigue (5.3 ± 0.5 vs. 5.1 ± 0.6 min). However, the relative decrease in the BCECF fluorescence ratio (and therefore pHi) from rest to the fatigue time point was significantly greater ( P < 0.05) during the first contractile period (to 65 ± 4% of initial resting values) compared with the second (77 ± 4%). The results of the present study demonstrated that, when preceded by an initial fatiguing contractile period, the rise in cytosolic H+ concentration in contracting single skeletal muscle fibers during a second contractile period was significantly reduced but did not attenuate the fatigue process in the second contractile period. These results suggest that intracellular factors other than H+ accumulation contribute to the fall in force development under these conditions.


1998 ◽  
Vol 274 (6) ◽  
pp. C1608-C1615 ◽  
Author(s):  
Philip A. Wahr ◽  
J. David Johnson ◽  
Jack. A. Rall

The influences of sarcomere uniformity and Ca2+ concentration on the kinetics of relaxation were examined in skinned frog skeletal muscle fibers induced to relax by rapid sequestration of Ca2+ by the photolysis of the Ca2+ chelator, diazo-2, at 10°C. Compared with an intact fiber, diazo-2-induced relaxation exhibited a faster and shorter initial slow phase and a fast phase with a longer tail. Stabilization of the sarcomeres by repeated releases and restretches during force development increased the duration of the slow phase and slowed its kinetics. When force of contraction was decreased by lowering the Ca2+concentration, the overall kinetics of relaxation was accelerated, with the slow phase being the most sensitive to Ca2+ concentration. Twitchlike contractions were induced by photorelease of Ca2+ from a caged Ca2+ (DM-Nitrophen), with subsequent Ca2+ sequestration by intact sarcoplasmic reticulum or Ca2+ rebinding to caged Ca2+. These twitchlike responses exhibited relaxation kinetics that were about twofold slower than those observed in intact fibers. Results suggest that the slow phase of relaxation is influenced by the degree of sarcomere homogeneity and rate of Ca2+ dissociation from thin filaments. The fast phase of relaxation is in part determined by the level of Ca2+ activation.


1986 ◽  
Vol 87 (2) ◽  
pp. 289-303 ◽  
Author(s):  
P Volpe ◽  
G Salviati ◽  
A Chu

The action of ruthenium red (RR) on Ca2+ loading by and Ca2+ release from the sarcoplasmic reticulum (SR) of chemically skinned skeletal muscle fibers of the rabbit was investigated. Ca2+ loading, in the presence of the precipitating anion pyrophosphate, was monitored by a light-scattering method. Ca2+ release was indirectly measured by following tension development evoked by caffeine. Stimulation of the Ca2+ loading rate by 5 microM RR was dependent on free Ca2+, being maximal at pCa 5.56. Isometric force development induced by 5 mM caffeine was reversibly antagonized by RR. IC50 for the rate of tension rise was 0.5 microM; that for the extent of tension was 4 microM. RR slightly shifted the steady state isometric force/pCa curve toward lower pCa values. At 5 microM RR, the pCa required for half-maximal force was 0.2 log units lower than that of the control, and maximal force was depressed by approximately 16%. These results suggest that RR inhibited Ca2+ release from the SR and stimulated Ca2+ loading into the SR by closing Ca2+-gated Ca2+ channels. Previous studies on isolated SR have indicated the selective presence of such channels in junctional terminal cisternae.


1999 ◽  
Vol 91 (1) ◽  
pp. 179-186 ◽  
Author(s):  
Gudrun Kunst ◽  
Bernhard M. Graf ◽  
Rupert Schreiner ◽  
Eike Martin ◽  
Rainer H. A. Fink

Background Although malignant hyperthermia after application of sevoflurane has been reported, little is known about its action on intracellular calcium homeostasis of skeletal muscle. The authors compared the effect of sevoflurane with that of isoflurane and halothane on Ca2+ release of mammalian sarcoplasmic reticulum and applied a novel method to quantify Ca2+ turnover in permeabilized skeletal muscle fibers. Methods Liquid sevoflurane, isoflurane, and halothane at 0.6 mM, 3.5 mM, and 7.6 mm were diluted either in weakly calcium buffered solutions with no added Ca2+ (to monitor Ca2+ release) or in strongly Ca2+ buffered solutions with [Ca2+] values between 3 nM and 24.9 microm for [Ca+]-force relations. Measurements were taken on single saponin skinned muscle fiber preparations of BALB/c mice. Individual [Ca2+]force relations were characterized by the Ca2+ concentration at half-maximal force that indicates the sensitivity of the contractile proteins and by the steepness. Each force transient was transformed directly into a Ca2+ transient with respect to the individual [Ca2+]-force relation of the fiber. Results At 0.6 mM, single force transients induced by sevoflurane were lower compared with equimolar concentrations of isoflurane and halothane (P &lt; 0.05). Similarly, calculated peak Ca2+ transients of sevoflurane were lower than those induced by equimolar halothane (P &lt; 0.05). The Ca2+ concentrations at half maximal force were decreased after the addition of sevoflurane, isoflurane, and halothane in a concentration-dependent manner (P &lt; 0.05). Conclusion Whereas sevoflurane, isoflurane, and halothane similarly increase the Ca2+ sensitivity of the contractile apparatus in skeletal muscle fibers, 0.6 mM sevoflurane induces smaller Ca2+ releases from the sarcoplasmic reticulum than does equimolar halothane.


1997 ◽  
Vol 73 (4) ◽  
pp. 1999-2011 ◽  
Author(s):  
P. Szentesi ◽  
Z. Papp ◽  
G. Szücs ◽  
L. Kovács ◽  
L. Csernoch

1986 ◽  
Vol 87 (2) ◽  
pp. 271-288 ◽  
Author(s):  
P Volpe ◽  
E W Stephenson

Isometric force and 45Ca efflux from the sarcoplasmic reticulum were measured at 19 degrees C in frog skeletal muscle fibers skinned by microdissection. After Ca2+ loading, application of the ionophores monensin, an Na+(K+)/H+ exchanger, or gramicidin D, an H+ greater than K+ greater than Na+ channel-former, evoked rapid force development and stimulated release of approximately 30% of the accumulated 45Ca within 1 min, whereas CCCP (carbonyl cyanide pyruvate p-trichloromethoxyphenylhydrazone), a protonophore, and valinomycin, a neutral, K+-specific ionophore, did not. When monensin was present in all bathing solutions, i.e., before and during Ca2+ loading, subsequent application failed to elicit force development and to stimulate 45Ca efflux. 5 min pretreatment of the skinned fibers with 50 microM digitoxin, a permeant glycoside that specifically inhibits the Na+,K+ pump, inhibited monensin and gramicidin D stimulation of 45Ca efflux; similar pretreatment with 100 microM ouabain, an impermeant glycoside, was ineffective. Monensin stimulation of 45Ca efflux was abolished by brief pretreatment with 5 mM EGTA, which chelates myofilament-space calcium. These results suggest that: monensin and gramicidin D stimulate Ca2+ release from the sarcoplasmic reticulum that is mediated by depolarization of the transverse tubules, which seal off after sarcolemma removal and form closed compartments; a transverse tubule membrane potential (myofilament space-negative) is maintained and/or established by the operation of the Na+,K+ pump in the transverse tubule membranes and is sensitive to the permeant inhibitor digitoxin; the transverse tubule-mediated stimulation of 45Ca efflux appears to be entirely Ca2+ dependent.


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