Intact Single Fibres Research Articles

Interference fine structure and sarcomere length dependence of the axial x-ray pattern from active single muscle fibers.

Axial x-ray diffraction patterns from single intact fibers of frog skeletal muscle were recorded by using a highly collimated x-ray beam at the European Synchrotron Radiation Facility. During isometric contraction at sarcomere lengths 2.2-3.2 microm, the M3 x-ray reflection, associated with the repeat of myosin heads along the filaments, was resolved into two peaks. The total M3 intensity decreased linearly with increasing sarcomere length and was directly proportional to the degree of overlap between myosin and actin filaments, showing that it comes from myosin heads in the overlap region. The separation between the M3 peaks was smaller at longer sarcomere length and was quantitatively explained by x-ray interference between myosin heads in the two overlap regions of each sarcomere. The relative intensity of the M3 peaks was independent of sarcomere length, showing that the axial periodicities of the nonoverlap and overlap regions of the myosin filament have the same value, 14.57 nm, during active contraction. In resting fibers the periodicity is 14.34 nm, so muscle activation produces a change in myosin filament structure in the nonoverlap as well as the overlap part of the filament. The results establish x-ray interferometry as a new tool for studying the motions of myosin heads during muscle contraction with unprecedented spatial resolution.

Is creatine kinase responsible for fatigue? Studies of isolated skeletal muscle deficient in creatine kinase.

Creatine kinase (CK) is a key enzyme for maintaining a constant ATP/ADP ratio during rapid energy turnover. To investigate the role of CK in skeletal muscle fatigue, we used isolated whole muscles and intact single fibers from CK-deficient mice (CK(-/-)). With high-intensity electrical stimulation, single fibers from CK(-/-) mice displayed a transient decrease in both tetanic free myoplasmic [Ca(2+)] ([Ca(2+)](i), measured with the fluorescent dye indo-1) and force that was not observed in wild-type fibers. With less intense, repeated tetanic stimulation single fibers and EDL muscles, both of which are fast-twitch, fatigued more slowly in CK(-/-) than in wild-type mice; on the other hand, the slow-twitch soleus muscle fatigued more rapidly in CK(-/-) mice. In single wild-type fibers, tetanic force decreased and [Ca(2+)](i) increased during the first 10 fatiguing tetani, but this was not observed in CK(-/-) fibers. Fatigue was not accompanied by phosphocreatine breakdown and accumulation of inorganic phosphate in CK(-/-) muscles. In conclusion, CK is important for avoiding fatigue at the onset of high-intensity stimulation. However, during more prolonged stimulation, CK may contribute to the fatigue process by increasing the myoplasmic concentration of inorganic phosphate.

Myosin isoforms in anuran skeletal muscle: their influence on contractile properties and in vivo muscle function.

Functional studies on isolated single anuran skeletal muscle cells represent classic experiments from which much of our understanding of muscle contraction mechanisms have been derived. Because of their superb mechanical stability when isolated, single anuran fibers provide a uniquely powerful model system that can be exploited to understand the relationship between myosin heavy chain (MHC) and myosin light chain (MLC) composition and muscle fiber function. In this review, we summarize historic and recent studies of MHC and MLC expression patterns in the fiber types of anuran species. We extend the traditional classification scheme, using data from recent reports in which frog MHCs have been cloned, to reveal the molecular basis of frog muscle fiber types. The influence of MHC and MLC isoforms on contractile kinetics of single intact fibers is reviewed. In addition, we discuss more subtle questions such as variability of myosin coexpression along a single cell, and its potential influence on contractile function. The frog jump is used as a model system to elucidate principles of muscular system design, including the role of MHC isoforms on in vivo muscle function. Sequence information is used from cloned frog MHCs to understand the role of specific regions of the myosin motor domain in regulating contractile function and the evolutionary origins of fast and slow amphibian MHCs. Finally, we offer promising future possibilities that combine molecular methods (such as recombinant gene transfer) with single cell contractile measurements to address questions regarding myosin structure/function and gene regulation.

Different effects of two gold compounds on muscle contraction, membrane potential and ryanodine receptor

Effects of gold sodium thiomalate and NaAuCl 4 on skeletal muscle function were studied using intact single fibres of frog skeletal muscle and fragmented sarcoplasmic reticulum prepared from frog and rabbit skeletal muscles. Gold sodium thiomalate at a concentration of 500 μM decreased tension amplitude by 27% and resting membrane potential by 5.3% after 30 and 22 min, respectively. The duration of tetanus tension was markedly shortened by 500 μM gold sodium thiomalate. When 10 μM NaAuCl 4 was applied to gold sodium thiomalate-pretreated fibres, the fibres lost the ability to contract upon electrical stimulation, similar to the effects of 10 μM NaAuCl 4 alone. In the presence of thiomalic acid, on the other hand, NaAuCl 4 did not completely block tetanus tension even at 50 μM. Thiomalic acid also inhibited NaAuCl 4-induced membrane depolarization. These findings suggest that thiomalate masks the effects of gold ion on muscle function. When sarcoplasmic reticulum vesicles were incorporated into lipid bilayers, exposure of the cis side of the Ca 2+-release channel to 100 μM gold sodium thiomalate rapidly increased the open probability of the channel 3.3-fold, from 0.032 in controls to 0.105, with an increase in number of open events and a decrease in mean closed time. The ability of NaAuCl 4 to activate the Ca 2+-release channel was much stronger than that of gold sodium thiomalate. Only 1 μM NaAuCl 4 was enough to activate the channel and this gold was effective from either side of the channel. These results suggest that gold sodium thiomalate could be used as an antirheumatic drug without considering severe side-effects on skeletal muscle. Coexistent thiomalate probably contributes to protection of muscle function from side-effects of gold ion.

MYOSIN HEAVY CHAIN AND LIGHT CHAIN VARIATIONS WITHIN AND BETWEEN SINGLE FROG MUSCLE FIBERS

1050 The myosin molecule in skeletal muscle is a hexamer composed of two myosin heavy chains (MHC) and four myosin light chains (MLC). These proteins have been demonstrated in skinned mammalian muscle fibers to have a significant effect on force-velocity properties1,2. MHC and MLC effects in intact single fibers are not as well characterized, because the myosin isoforms are not as well defined. We identified the MHCs within frog muscle3 and now report correlations between MHCs and MLCs. Isolated fibers (n = 65) from hindlimb muscles of Rana pipiens were solublized in SDS buffer for analysis of MHCs and MLCs on SDS-PAGE gels. Quantitative analysis of each fiber demonstrated a significant but very weak linear correlation between the MLC3/MLC1 ratio and percent type 1 MHC (r2 = 0.12, p<0.005). Dramatic variations in both MHC (n=7) and MLC (n=6) occurred between 1 mm segments along the length of fibers. In the most extreme case, type 2 MHC isoform decreased from 100% of the total MHC pool at the fiber ends to 50% in the center segments. MLC3/MLC1 ratio varied by 2.7-fold between segments. There was no clear correlation between the variability in MHC isoforms and MLC3/MLC1 ratio along the fiber length, implying that these protein subunits are not strictly coregulated This work was supported by NIH grant AR40050 and the VA.

Z-line structural diversity in frog single muscle fiber in the passive state.

The structural changes of the Z-line between small square net (ss) and basket weave (bw) cross-sectional patterns were examined using intact single fibers and mechanically skinned fibers in the passive state to determine if the pattern is related to the sarcomere length (SL) and if the pattern undergoes a reversible transition in low- and high-osmotic medium. Frog single fibers were isolated from the anterior tibial muscle in Ringer's solution. Entirely or partially skinned single fibers were prepared in relaxing solution (also called low-osmotic medium). The high osmotic medium contained 10% polyvinylpyrrolidone (PVP) in relaxing solution. The sarcomere length (SL) of each fiber was measured directly by use of a laser beam or indirectly from electron micrographs with use of a correction factor. The ss and bw forms in cross sections were quantified by analysis of electron micrographs. The results show that the structural change of Z-line occurs around bw << 2.3-2.4 microns << ss (n = 25) and bw << 3.1-3.2 microns << ss (n = 13) in intact single fibers and skinned fibers, respectively. With the quick freeze-freeze substitution method, an intact single fiber with a SL of 2.35 microns showed almost 100% of ss form. The structural transition in cross section was also confirmed in four partially skinned fibers, where patterns went from mostly ss form (intact portion) to mostly bw form (skinned portion) at the SL between 2.40 to 3.20 microns. The reversibility of the change between ss and bw was proved by using low- and high-osmotic medium. The transition and reversion of cross-sectional patterns both occur in the passive state.

Effect of hydrogen peroxide and dithiothreitol on contractile function of single skeletal muscle fibres from the mouse.

1. We used intact single fibres from a mouse foot muscle to study the role of oxidation-reduction in the modulation of contractile function. 2. The oxidant hydrogen peroxide (H2O2, 100-300 microM) for brief periods did not change myoplasmic Ca2+ concentrations ([Ca2+]i) during submaximal tetani. However, force increased by 27 % during the same contractions. 3. The effects of H2O2 were time dependent. Prolonged exposures resulted in increased resting and tetanic [Ca2+]i, while force was significantly diminished. The force decline was mainly due to reduced myofibrillar Ca2+ sensitivity. There was also evidence of altered sarcoplasmic reticulum (SR) function: passive Ca2+ leak was increased and Ca2+ uptake was decreased. 4. The reductant dithiothreitol (DTT, 0.5-1 mM) did not change tetanic [Ca2+]i, but decreased force by over 40 %. This was completely reversed by subsequent incubations with H2O2. The force decline induced by prolonged exposure to H2O2 was reversed by subsequent exposure to DTT. 5. These results show that the elements of the contractile machinery are differentially responsive to changes in the oxidation-reduction balance of the muscle fibres. Myofibrillar Ca2+ sensitivity appears to be especially susceptible, while the SR functions (Ca2+ leak and uptake) are less so.

Evidence for Na+/Ca2+ exchange in intact single skeletal muscle fibers from the mouse.

The myoplasmic free Ca2+ concentration ([Ca2+]i) was measured in intact single fibers from mouse skeletal muscle with the fluorescent Ca2+ indicator indo 1. Some fibers were perfused in a solution in which the concentration of Na+ was reduced from 145.4 to 0.4 mM (low-Na+ solution) in an attempt to activate reverse-mode Na+/Ca2+ exchange (Ca2+ entry in exchange for Na+ leaving the cell). Under normal resting conditions, application of low-Na+ solution only increased [Ca2+]i by 5.8 +/- 1.8 nM from a mean resting [Ca2+]i of 42 nM. In other fibers, [Ca2+]i was elevated by stimulating sarcoplasmic reticulum (SR) Ca2+ release with caffeine (10 mM) and by inhibiting SR Ca2+ uptake with 2,5-di(tert-butyl)-1,4-benzohydroquinone (TBQ; 0.5 microM) in an attempt to activate forward-mode Na+/Ca2+ exchange (Ca2+ removal from the cell in exchange for Na+ influx). These two agents caused a large increase in [Ca2+]i, which then declined to a plateau level approximately twice the baseline [Ca2+]i over 20 min. If the cell was allowed to recover between exposures to caffeine and TBQ in a solution in which Ca2+ had been removed, the increase in [Ca2+]i during the second exposure was very low, suggesting that Ca2+ had left the cell during the initial exposure. Application of caffeine and TBQ to a preparation in low-Na+ solution produced a large, sustained increase in [Ca2+]i of approximately 1 microM. However, when cells were exposed to caffeine and TBQ in a low-Na+ solution in which Ca2+ had been removed, a sustained increase in [Ca2+]i was not observed, although [Ca2+]i remained higher and declined slower than in normal Na+ solution. This suggests that forward-mode Na+/Ca2+ exchange contributed to the fall of [Ca2+]i in normal Na+ solution, but when extracellular Na+ was low, a prolonged elevation of [Ca2+]i could activate reverse-mode Na+/Ca2+ exchange. The results provide evidence that skeletal muscle fibers possess a Na+/Ca2+ exchange mechanism that becomes active in its forward mode when [Ca2+]i is increased to levels similar to that obtained during contraction.

AM-loading of fluorescent Ca2+ indicators into intact single fibers of frog muscle

The AM loading of a number of different fluorescent Ca2+ indicators was compared in intact single fibers of frog muscle. Among the 13 indicators studied, loading rates (the average increase in the fiber concentration of indicator per first 60 min of loading) varied approximately 100-fold, from approximately 3 microM/h to >300 microM/h (16 degrees C). Loading rates were strongly dependent on the molecular weight of the AM compounds, with the rate increasing steeply as molecular weight decreased below approximately 850. Properties of delta F/F (the Ca2(+)-related fluorescence signal observed with fiber stimulation) were also measured in AM-loaded fibers and compared with those previously reported for fibers microinjected with indicator. In general, the time course of delta F/F was very similar with AM-loading and microinjection; however, the amplitude of delta F/F was usually smaller with AM-loading. There was a strong correlation between the rate of indicator loading and the value of the parameter f (the ratio of the amplitude of delta F/F in AM-loaded versus microinjected fibers). For indicators with small loading rates (<10 microM/h, N = 5), f values were generally small (< or =0.4, N = 4); whereas with large loading rates (>100 microM/h, N = 4), f values were large (> or =0.8, N = 4). This suggests that, with any AM indicator, a small concentration may associate nonspecifically with the fiber (either the indicator is incompletely de-esterified or, if completely de-esterified, not located in the myoplasmic compartment). If the loaded concentration is small, the nonspecific indicator will present a significant source of error in the estimation of [Ca2+]i.

The effects of glibenclamide on tetanic force and intracellular calcium in normal and fatigued mouse skeletal muscle

In this study the effects of ATP-sensitive K+ channel modulators were studied in intact single fibres dissected from mouse skeletal muscle. Indo-1 was used to measure [Ca2+]i simultaneously with force during normal and fatiguing stimulation. In control fibres, opening of ATP-sensitive K+ channels with BRL 38227 produced a small reduction in tetanic force and [Ca2+]i. In contrast, glibenclamide, a selective blocker of the ATP-sensitive K+ channel, slightly increased tetanic force and [Ca2+]i in these fibres and also increased Ca2+ sensitivity. Glibenclamide produced a more marked increase in tetanic force and [Ca2+]i during the later stages of fatiguing stimulation, although this effect was observed in only 50% of fibres examined. We conclude from this study that glibenclamide produces a partial reversal of the later stages of fatigue in a subpopulation of muscle fibres. Opening of ATP-sensitive K+ channels may therefore contribute to the decline in tetanic force and [Ca2+]i characteristic of skeletal muscle fatigue.

Calcium transients in intact rat skeletal muscle fibers in agarose gel.

Intact single fibers enzymatically dissociated from rat flexor digitorum brevis muscle were suspended in 0.5% low-melting-temperature agarose gel to minimize fiber movement during action potentials or trains of action potentials. Resting Ca2+ concentration ([Ca2+]) and changes in [Ca2+] were monitored using the fluorescent calcium indicator fura 2. The time course and waveform of [Ca2+] transients during an action potential or trains of action potentials in fibers in agarose were calculated using kinetic parameters previously determined to correct for the calcium-fura 2 kinetic delay. Half times of the calculated calcium transients for single action potentials were 30-fold briefer than the original fura 2 signals. To confirm the time course and waveform of the calculated calcium transients, changes in [Ca2+] were monitored using the more rapidly equilibrating calcium indicator mag-fura 2. [Ca2+] transients for fibers containing fura 2 had very similar time courses and waveforms as mag-fura 2 signals from other fibers, indicating that the corrections for the calcium-fura 2 kinetic delay were accurate. The advantages of the agarose gel suspension are discussed.

Force‐velocity relation for frog muscle fibres: effects of moderate fatigue and of intracellular acidification.

1. Intact frog single fibres were investigated under control conditions (1 s tetanus every 2, 3 or 5 min) and during moderate fatigue (interval between tetani 15 or 30 s). 2. Fatigue reduced isometric force (P0) by 25.8 +/- 1.6% (S.E.M.; n = 13) and depressed the maximum velocity of shortening (Vmax) by 10.2 +/- 2.2% (n = 13). The force-velocity relation became less curved, a/P0* (see Methods) being increased by 29.5 +/- 8.8% (n = 13). Thus, power was less affected than isometric force or Vmax. 3. The velocity of unloaded shortening (V0), from slack test measurements, was reduced proportionally more than Vmax during fatigue. Under control conditions V0 was larger than Vmax, but during fatigue their values were not significantly different. 4. Stiffness during shortening was reduced during fatigue indicating fewer attached cross-bridges in fatigue. Force was reduced more than stiffness indicating that, on average, there is less force per attached cross-bridge. 5. The force-lengthening velocity relation showed that the ability to resist forces greater than isometric was well preserved in fatigue. 6. Compared with fatigue, intracellular acidification with CO2 produced a smaller reduction in isometric force. However, reduction in Vmax was not significantly different from that in fatigue. These results are consistent with both inorganic phosphate and H+ increasing in fatigue, but only H+ increasing during acidification, and isometric force being reduced by both, Vmax being sensitive only to H+.

Birefringence changes associated with isometric contraction and rapid shortening steps in frog skeletal muscle fibres.

1. Muscle birefringence, the difference between the refractive indices of light polarized parallel and perpendicular to the muscle fibre axis, was measured at 3 degrees C in intact single fibres isolated from frog muscle. Resting birefringence was 2.20 +/- 0.02 x 10(-3) (mean +/- S.E.M., n = 44) at sarcomere length 2.4-2.7 microns and 2.35 +/- 0.03 x 10(-3) (n = 19) at 3.5-3.8 microns. 2. Birefringence decreased during isometric twitch or tetanic contractions. The peak change in a twitch at sarcomere length 2.6 microns, determined by two independent methods, was 0.150 +/- 0.017 x 10(-3) (mean +/- S.E.M., n = 6). The corresponding value after 0.4 s of tetanic stimulation was 0.167 +/- 0.012 x 10(-3) (n = 6). 3. The birefringence change had a shorter latency than tension and reached its half-maximum value earlier than tension. The difference in time to half-maximum in tetani was 11.5 +/- 1.3 ms (mean +/- S.E.M., n = 6) at 3 degrees C. After stimulation birefringence recovered to its pre-stimulus baseline more slowly than tension. 4. The birefringence decrease after 0.4 s of tetanic stimulation was linearly related to the expected degree of overlap between actin and myosin filaments in the sarcomere length range 2.6-3.6 microns. The amplitude of the birefringence decrease at full filament overlap (sarcomere length 2.2 microns) was estimated to be 0.235 +/- 0.015 x 10(-3). 5. Birefringence changes associated with shortening steps of 0.9% fibre length at sarcomere length 2.6 microns exhibited four phases corresponding to those of the tension transient. There was no consistent birefringence change during the length step itself. During the rapid tension recovery birefringence increased by 0.014 +/- 0.001 x 10(-3) (n = 3), measured from the end of the length step to 2 ms later. Birefringence continued to increase as tension recovery slowed, reaching a peak about 10 ms after the step, then recovered with a rate similar to that of the final tension recovery. 6. These birefringence changes are likely to be caused by axial rotation of the head domain of the myosin cross-bridge. During isometric contraction heads bind to actin with their long axes more perpendicular to the fibre axis than in resting muscle, although there is likely to be a wide range of head orientations during contraction.(ABSTRACT TRUNCATED AT 400 WORDS)

Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3

Fluo-3 is an unusual tetracarboxylate Ca2+ indicator. For recent lots supplied by Molecular Probes Inc. (Eugene, OR), FMAX, the fluorescence intensity of the indicator in its Ca(2+)-bound form, is approximately 200 times that of FMIN, the fluorescence intensity of the indicator in its Ca(2+)-free form. (For earlier lots, impurities may account for the smaller reported values of FMAX/FMIN, 36-40). We have injected fluo-3 from a high-purity lot into intact single fibers from frog muscle and measured the indicator's absorbance and fluorescence signals at rest (A and F, respectively) and changes in absorbance and fluorescence following action potential stimulation (delta A and delta F signals substantially lagged behind that of the myoplasmic free Ca2+ transient. Our analysis of fluo-3's signals from myoplasm therefore focused on information about the level of resting myoplasmic free [Ca2+] ([Ca2+]r). From A, delta A, and in vitro estimates of fluo-3's molar extinction coefficients, the change in the fraction of fluo-3 in the Ca(2+)-bound form during activity (delta f) was estimated. From delta f, delta F, and F, the fraction of the indicator in the Ca(2+)-bound form in the resting fiber (fr) was estimated by fr = (delta f x F/delta F) + (1-FMAX/FMIN)-1. Since FMAX/FMIN is large, the contribution of the second term to the estimate of fr is small. At 16 degrees C, the mean value (mean +/- S.E.) of fr was 0.086 +/- 0.004 (N = 15). From two estimates of the apparent dissociation constant of fluo-3 for Ca2+ in the myoplasm, 1.09 and 2.57 microM, the average value of [Ca2+]r is calculated to be 0.10 and 0.24 microM, respectively. The smaller of these estimates lies near the upper end of the range of values for [Ca2+]r in frog fibers (0.02-0.12 microM) estimated by others with aequorin and Ca(2+)-selective electrodes. The larger of the estimates lies within the range of values (0.2-0.3 microM) previously estimated in this laboratory with fura red. We conclude that [Ca2+]r in frog fibers is at least 0.1 microM and possibly as large as 0.3 microM.

The influence of intracellular pH on contraction, relaxation and [Ca2+]i in intact single fibres from mouse muscle.

1. The effects of intracellular pH (pHi) on myoplasmic free calcium concentration ([Ca2+]i) and contractile performance were studied in intact single fibres dissected from mouse skeletal muscle. Indo-1 was used to measure [Ca2+]i and pHi was altered by changing perfusate CO2. 2. Tetanic tension was decreased at acidic pHi and increased at alkaline pHi whereas the rate of mechanical relaxation was showed at both acidic and alkaline pHi. Resting and tetanic [Ca2+]i were increased at acidic pHi and decreased at alkaline pHi while the final rate of decline of [Ca2+]i after a tetanus was markedly slowed at acid pHi but only marginally accelerated at alkaline pHi. 3. Steady-state [Ca2+]i-tension curves were constructed from measurements of tetani at different stimulus frequencies. The curves at acid pHi showed a reduced maximum Ca(2+)-activated tension and a reduced Ca2+ sensitivity, and curves at alkaline pHi showed the opposite changes. 4. Two methods were devised to determine the contribution of [Ca2+]i to the rate of relaxation. In one method the instantaneous tension was plotted as a function of instantaneous [Ca2+]i throughout a tetanus and compared with the steady-state [Ca2+]i-tension relation. In a second method the [Ca2+]i signal during a tetanus was converted to a Ca(2+)-derived tension record by means of the steady-state [Ca2+]i-tension relation and this Ca(2+)-derived tension was then compared to the true tension. 5. The sarcoplasmic reticulum (SR) pump function was analysed by plotting -d[Ca2+]i/dt against [Ca2+]i during the final slow decline of [Ca2+]i after a tetanus. This analysis shows that the Ca2+ uptake by the SR is a third- or fourth-power function of [Ca2+]i and that acidosis substantially slows the rate of SR Ca2+ pumping. 6. In conclusion, the slowing of relaxation at acidic pHi could be attributed to a slowing of cross-bridge detachment rather than the observed slowing of the rate of uptake of Ca2+. Conversely the slowing of relaxation in alkaline pHi could be attributed to the increase of Ca2+ sensitivity of the myofibrillar proteins.

Effects of 2,3-butanedione monoxime on the crossbridge kinetics in frog single muscle fibres.

The effects of 2,3-butanedione monoxime (BDM) on contraction characteristics were studied at 5 degrees C in single intact fibres isolated from the tibialis anterior muscle of the frog. The force-velocity relation was determined using the controlled-velocity method in either whole fibres or short fibre segments in which sarcomere shortening was measured by a laser light diffraction method. It is shown that 3 mM BDM decreases the speed of rise and the amount of tetanus tension, reduces the maximum velocity of shortening and increases the curvature of the force-velocity relation, as well as the value for the stiffness to tension ratio. BDM also slowed down the redevelopment of tetanus tension after a period of unloaded shortening both in fixed-end and in length-clamp conditions. In normal and in BDM-treated fibres length-clamping increased the speed of the initial rise of tetanus tension but not that of the recovery after shortening. The observed force-velocity data points were fitted by the Huxley (1957) equation. It was found that BDM produces a conspicuous decrease of the rate constant for crossbridge attachment. This effect, and also a reduction of the force per crossbridge, are responsible for the depression of the contractile characteristics produced by BDM.

Myoplasmic free Mg2+ concentration during repetitive stimulation of single fibres from mouse skeletal muscle.

1. The role of the myoplasmic free Mg2+ concentration ([Mg2+]i) in fatigue was studied in intact single fibres isolated from mouse skeletal muscle. Fatigue was produced by repeated tetanic stimulation. The fluorescent Mg2+ indicator furaptra was pressure injected into fibres. In vivo calibrations were performed to convert fluorescence signals into [Mg2+]i. 2. [Mg2+]i at rest was 0.78 +/- 0.05 mM (mean +/- S.E.M., n = 14). An increase of the extracellular [Mg2+] from 0.5 to 20 mM resulted in a small elevation of [Mg2+]i (86 microM in 5 min). Removal of extracellular Na+ did not affect [Mg2+]i. An intracellular alkanization of about 0.6 pH units gave a [Mg2+]i reduction of 65 microM. 3. During fatiguing stimulation [Mg2+]i initially remained almost constant and it then suddenly started to rise towards the end of the stimulation period. The onset of the [Mg2+]i rise was always followed by a rapid tension decline. In fatigue [Mg2+]i was approximately twice as high as at rest. 4. Fibres were injected with MgCl2 to study if the rise in [Mg2+]i could explain the tension decline in fatigue. An elevation of [Mg2+]i was accompanied by a tension reduction but the [Mg2+]i for a given tension was generally much higher in rested fibres injected with MgCl2 than in fatigued fibres. Thus the rise in [Mg2+]i as such cannot explain the tension reduction in fatigue. 5. Injection of MgCl2 was also used to assess the intracellular Mg2+ buffering. The mean Mg2+ buffer power (i.e. the ratio of the change in [Mg2+]i to the amount of Mg2+ added) was 0.62. 6. ATP is the quantitatively most important binding site for Mg2+ at rest and ATP breakdown is then a likely source of the [Mg2+]i increase in fatigue. The role of ATP breakdown in the increase of [Mg2+]i was studied with metabolic inhibition: fibres were exposed to iodoacetic acid to inhibit glycolysis and cyanide to inhibit oxidative phosphorylation. The pattern during metabolic inhibition was similar to that observed during fatigue. After remaining almost constant during a lengthy period, [Mg2+]i rose rapidly and this rise preceded a period of rapid tension decline. The fibres thereafter went into rigor and [Mg2+]i stabilized at an elevated level; the mean [Mg2+]i increase in rigor was 1.30 mM. 7. We have used modelling to determine the likely change in the intracellular ATP concentration ([ATP]i) for the observed changes in [Mg2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple actions of 2,3‐butanedione monoxime on contractile activation in frog twitch fibres.

1. The effects of 2,3-butanedione monoxime (BDM) on various steps in the excitation-contraction coupling sequence, including action potential, charge movement and twitch tension, were studied in twitch fibres of Rana temporaria. 2. The resting potential of intact fibres in whole muscle bathed in 20 mM-BDM was the same as control. The resting potential also remained stable after more than 100 min in 20 mM-BDM. 3. The action potential was measured in intact fibres of fibre bundles with an intracellular microelectrode. Applications of 5 and 7.5 mM-BDM had no effect on its amplitude, whereas 10 and 20 mM suppressed its amplitude by about 4 and 10%, respectively. Increasing concentrations of BDM prolonged the half-width and elevated the after-potential of the action potential progressively. The action potential was also measured in cut fibres mounted in a double Vaseline-gap chamber. Results were similar to those in intact fibres. 4. Charge movement was measured in intact fibres of halved muscles with the three-microelectrode voltage-clamp technique. The steady-state Q-V plot of the total charge measured in isotonic tetraethylammonium (TEA) Ringer solution with 20 mM-BDM appeared to be shifted about 10 mV in the depolarizing direction and to be slightly more shallow when compared with the control Q-V plot measured in hypertonic TEA Ringer solution with 350 mM-sucrose. After allowing for the voltage shift, 20 mM-BDM did not appear to affect the kinetics of both components of charge movement, but suppressed the maximum amount of total charge by about one-quarter. 5. Charge movement was also measured in cut fibres with the double Vaseline-gap voltage-clamp technique. In the presence of 20 mM-BDM, charge movement traces resembled those from intact fibres. Twenty millimolar BDM suppressed the maximum amount of total charge by about one-quarter, as in intact fibres. The steady-state Q-V plots from cut fibres were separated into Q beta (early current) and Q gamma (late hump current) components by least-squares fitting with a sum of two Boltzmann distribution functions. On average, 20 mM-BDM suppressed Q beta and Q gamma in roughly equal proportion, but did not affect the individual voltage distributions of Q beta and Q gamma. 6. Twitch tension was measured in single intact fibres stimulated extracellularly. BDM effectively reduced the peak amplitude, the time-to-peak and the half-width of twitch tension. The interaction of BDM with receptors appeared to follow more or less a simple 1:1 binding in fibres stretched to sarcomere lengths of about 3.6 microns.(ABSTRACT TRUNCATED AT 400 WORDS)

Myoplasmic calcium transients monitored with purpurate indicator dyes injected into intact frog skeletal muscle fibers.

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after microinjection with tetramethylmurexide (TMX) or purpurate-3,3' diacetic acid (PDAA), two compounds from the purpurate family of absorbance Ca2+ indicators previously used in cut muscle fibers (Maylie, J., M. Irving, N. L. Sizto, G. Boyarsky, and W. K. Chandler. 1987. J. Gen. Physiol. 89:145-176; Hirota, A., W. K. Chandler, P. L. Southwick, and A. S. Waggoner. 1989. J. Gen. Physiol. 94:597-631.) The apparent longitudinal diffusion constant of PDAA (mol wt 380) in myoplasm was 0.99 (+/- 0.04, SEM) x 10(-6) cm2 s-1 (16-17 degrees C), a value which suggests that 24-43% of the PDAA molecules were bound to myoplasmic constituents of large molecular weight. The corresponding values for TMX (mol wt 322) were 0.98 (+/- 0.05) x 10(-6) cm2 s-1 and 44-50%, respectively. Muscle membranes (surface and/or transverse-tubular) appear to be permeable to TMX and, to a lesser extent, to PDAA, since the total amount of indicator contained within a fiber decreased with time after injection. The average time constants for disappearance of indicator were 46 (+/- 7, SEM) min for TMX and 338 (+/- 82) min for PDAA. The fraction of indicator in the Ca2(+)-bound state in resting fibers was significantly different from zero for TMX (0.070 +/- 0.008) but not for PDAA (0.026 +/- 0.009). In in vitro calibrations PDAA but not TMX appeared to react with Ca2+ with 1:1 stoichiometry. In agreement with Hirota et al. (Hirota, A., W. K. Chandler, P. L. Southwick, and A. S. Waggoner. 1989. J. Gen. Physiol. 94:597-631), we conclude that PDAA is probably a more reliable myoplasmic Ca2+ indicator than TMX. In fibers that contained PDAA and were stimulated by a single action potential, the calibrated peak value of the myoplasmic free [Ca2+] transient (delta[Ca2+]) averaged 9.4 (+/- 0.6) microM, a value about fivefold larger than that calibrated with antipyrylazo III under otherwise identical conditions (Baylor, S. M., and S. Hollingworth. 1988. J. Physiol. 403:151-192). The fivefold difference is similar to that previously reported in cut fibers with antipyrylazo III and PDAA. Since in both intact and cut fibers the percentage of PDAA bound to myoplasmic constituents is considerably smaller than that found for antipyrylazo III, the PDAA calibration of delta[Ca2+] is likely to be more accurate. Interestingly, in intact fibers the peak value of delta[Ca2+] calibrated with either PDAA or antipyrylazo III is about half that calibrated in cut fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in phenol red absorbance in response to electrical stimulation of frog skeletal muscle fibers.

Intact single twitch fibers from frog muscle were stretched to long sarcomere length, micro-injected with the pH indicator dye phenol red, and activated by action potential stimulation. Indicator-related absorbance changes (denoted by delta A0 and delta A90) were measured with 0 degree and 90 degrees polarized light (oriented, respectively, parallel and perpendicular to the fiber axis). Two components of delta A were detected that had generally similar time courses. The "isotropic" component, calculated as the weighted average (delta A0 + 2 delta A90)/3, had the wavelength dependence expected for a change in myoplasmic pH. If calibrated in pH units, this signal's peak amplitude, which occurred 15-20 ms after stimulation, corresponded to a myoplasmic alkalization of average value 0.0025 +/- 0.0002 (+/- SEM; n = 9). The time course of this change, as judged from a comparison with that of the fibers' intrinsic birefringence signal, was delayed slightly with respect to that of the myoplasmic free [Ca2+] transient. On average, the times to half-peak and peak of the phenol red isotropic signal lagged those of the birefringence signal by 2.4 +/- 0.2 ms (+/- SEM; n = 8) and 8.4 +/- 0.5 ms (+/- SEM; n = 4), respectively. The other component of the phenol red signal was "dichroic," i.e., detected as a difference (delta A0-delta A90 greater than 0) between the two polarized absorbance changes. The wavelength dependence of this signal was similar to that of the phenol red resting dichroic signal (Baylor and Hollingworth. 1990. J. Gen. Physiol. 96:449-471). Because of the presence of the active dichroic signal, and because approximately 80% of the phenol red molecules appear to be bound in the resting state to either soluble or structural sites, the possibility exists that myoplasmic events other than a change in pH underlie the phenol red isotropic signal.