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.     

Simulation of calcium sparks in cut skeletal muscle fibers of the frog

Spark mass, the volume integral of Delta F/F, was investigated theoretically and with simulations. These studies show that the amount of Ca2+ bound to fluo-3 is proportional to mass times the total concentration of fluo-3 ([fluo-3T]); the proportionality constant depends on resting Ca2+ concentration ([Ca2+]R). In the simulation of a Ca2+ spark in an intact frog fiber with [fluo-3T] = 100 microM, fluo-3 captures approximately one-fourth of the Ca2+ released from the sarcoplasmic reticulum (SR). Since mass in cut fibers is several times that in intact fibers, both with similar values of [fluo-3T] and [Ca2+]R, it seems likely that SR Ca2+ release is larger in cut fiber sparks or that fluo-3 is able to capture a larger fraction of the released Ca2+ in cut fibers, perhaps because of reduced intrinsic Ca2+ buffering. Computer simulations were used to identify these and other factors that may underlie the differences in mass and other properties of sparks in intact and cut fibers. Our spark model, which successfully simulates calcium sparks in intact fibers, was modified to reflect the conditions of cut fiber measurements. The results show that, if the protein Ca2+-buffering power of myoplasm is the same as that in intact fibers, the Ca2+ source flux underlying a spark in cut fibers is 5-10 times that in intact fibers. Smaller source fluxes are required for less buffer. In the extreme case in which Ca2+ binding to troponin is zero, the source flux needs to be 3-5 times that in intact fibers. An increased Ca2+ source flux could arise from an increase in Ca2+ flux through one ryanodine receptor (RYR) or an increase in the number of active RYRs per spark, or both. These results indicate that the gating of RYRs, or their apparent single channel Ca2+ flux, is different in frog cut fibers--and, perhaps, in other disrupted preparations--than in intact fibers.     

Low myoplasmic Mg2+ potentiates calcium release during depolarization of frog skeletal muscle fibers.

The role of intracellular free magnesium concentration ([Mg2+]) in modulating calcium release from the sarcoplasmic reticulum (SR) was studied in voltage-clamped frog cut skeletal muscle fibers equilibrated with cut end solutions containing two calcium indicators, fura-2 and antipyrylazo III (AP III), and various concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor calcium transients, whereas fura-2 fluorescence was used to monitor resting calcium. The rate of release (Rrel) of calcium from the SR was calculated from the calcium transient and found to be increased in low internal [Mg2+]. After correcting for effects of calcium depletion from the SR and normalization to SR content, the mean values of the inactivatable and noninactivatable components of Rrel were increased by 163 and 46%, respectively, in low Mg2+. Independent of normalization to SR content, the ratio of inactivatable to noninactivatable components of Rrel was increased in low internal [Mg2+]. Both observations suggest that internal [Mg2+] preferentially modulates the inactivatable component of Rrel, which is thought to be due to calcium-induced calcium release from the SR. This could also explain the observation that, in low internal [Mg2+], the time to the peak of the calcium transient for a 5-ms depolarizing pulse was not very different from the time to the peak of the delta [Ca2+] for a 10-ms pulse of the same amplitude. Finally, in low internal [Mg2+], the calcium transient elicited by a short depolarizing pulse was in some cases clearly followed by a very slow rise of calcium after the end of the pulse. The observed effects of reduced [Mg2+] on calcium release are consistent with a removal of the inhibition that the normal 1 mM myoplasmic [Mg2+] exerts on calcium release in skeletal muscle fibers.     

Effects of low myoplasmic Mg2+ on calcium binding by parvalbumin and calcium uptake by the sarcoplasmic reticulum in frog skeletal muscle.

The effects of low intracellular free Mg2+ on the myoplasmic calcium removal properties of skeletal muscle were studied in voltage-clamped frog skeletal muscle fibers by analyzing the changes in intracellular calcium and magnesium due to membrane depolarization under various conditions of internal free [Mg2+]. Batches of fibers were internally equilibrated with cut end solutions containing two calcium indicators, antipyrylazo III (AP III) and fura-2, and different concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor [Ca2+] and [Mg2+] transients, whereas fura-2 fluorescence was mostly used to monitor resting [Ca2+]. Shortly after applying an internal solution containing less than 60 microM free Mg2+ to the cut ends of depolarized fibers most of the fibers exhibited spontaneous repetitive movements, suggesting that free internal Mg2+ might affect the activity of the sarcoplasmic reticulum (SR) calcium channels at rest. The spontaneous contractions generally subsided. In polarized fibers the maximal amplitude of the calcium transient elicited by a depolarizing pulse was about the same whatever the internal [Mg2+], but its decay after the end of the pulse slower in low [Mg2+]. In low [Mg2+] (less than 0.14 mM), the mean rate constant of decay obtained from fitting a single exponential plus a constant to the decay of the calcium transients was approximately 30% of its value in the control fibers (1 mM internal [Mg2+]). A model characterizing the main calcium removal properties of a frog skeletal muscle fiber, including the SR pump and the Ca-Mg sites on parvalbumin, was fitted to the decay of the calcium transients. Results of the fits show that in low internal [Mg2+] the slowing of the decay of the calcium transient can be well predicted by both a decreased rate of SR calcium uptake and an expected decreased resting magnesium occupancy of parvalbumin leading to a reduced contribution of parvalbumin to the overall rate of calcium removal. These results are thus consistent with the known properties of parvalbumin as a Ca-Mg buffer and furthermore suggest that in an intact portion of a muscle fiber, the activity of the SR calcium pump can be affected by the level of free Mg2+.     

Caffeine contracture and iodoacetate rigor in frog skeletal muscle. A comparison

Frog sartorius muscle treated with 5.0 mM or greater caffeine exhibits stiffness similar to that obtained from muscle in iodoacetate rigor. The data provide quantitative evidence that suggests that caffeine at irreversible contracture-producing concentrations somehow induces a rigor or rigorlike state in skeletal muscle.     

Minimal latency of calcium release in frog twitch muscle fibres

Intracellular release of calcium in frog skeletal muscle fibres was monitored by the use of arsenazo III, in response to voltage clamped depolarizing pulses. A latency of a few milliseconds was evident between the onset of depolarization and the first detectable rise in the arsenazo-calcium signal, and this decreased logarithmically as the depolarization was increased. The minimal latency with strong depolarization (to +20 to +100 mV) was about 2 ms at 5 degrees C. This delay appears to be sufficiently long to be compatible with a chemically mediated coupling mechanism between depolarization and calcium release from the sarcoplasmic reticulum.     

Caffeine treatment inhibits drug-induced calcium release from sarcoplasmic reticulum and caffeine contracture but not tetanus in frog skeletal muscle

Effects of pretreatment with caffeine on Ca2+ release induced by caffeine, thymol, quercetin, or p-chloromercuriphenylsulfonic acid (pCMPS) from the heavy fraction of sarcoplasmic reticulum (SR) were studied and compared with those effects on caffeine contracture and tetanus tension in single fibers of frog skeletal muscle. Caffeine (1-5 mM) did induce transient Ca2+ release from SR vesicles, but subsequent further addition of caffeine (10 mM, final concentration) induced little Ca2+ release. Ca2+ release induced by thymol, quercetin, or pCMPS was also inhibited by pretreatment with caffeine. In single muscle fibers, pretreatment with caffeine (1-5 mM) partially reduced the contracture induced by 10 mM caffeine. However, tetanus tension was almost maximally induced by electrical stimulus in caffeine-treated fibers. These results indicate that SR, which becomes less sensitive to caffeine, thymol, quercetin, or pCMPS by pretreatment with caffeine, can still respond to a physiological signal transmitted from transverse tubules.     

Determination of ionic calcium in frog skeletal muscle fibers

Ionic calcium concentrations were measured in frog skeletal muscle fibers using Ca-selective microelectrodes. In fibers with resting membrane potentials more negative than -85 mV, the mean pCa value was 6.94 (0.12 microM). In fibers depolarized to -73 mV with 10-mM K the mean pCa was 6.43 (0.37 microM). This increase in the intracellular [Ca2+] could be related to the higher oxygen consumption and heat production (Solandt effect) reported to occur under these conditions. Caffeine, 3 mM, also produced an increase in the free ionic calcium to a pCa of 6.52 (0.31 microM) without changes in the membrane potential. Lower caffeine concentrations, 1 and 2 mM, did not change the fiber pCa. Lower Ca concentrations in the external medium effectively reduced the internal ionic calcium to an estimated pCa of 7.43 (0.03 microM).     

Nystatin-, mycoheptin- and levorin-induced conductance in the membrane of frog skeletal muscle fibres

The effects of the polyene antibiotics nystatin (2 × 10^–5–10^–4 mol/l), mycoheptin (1.3 × 10^–6–10^–5 mol/l) and levorin (10^–8–5 × 10^–5 mol/l)on isolated frog skeletal muscle fibres and whole sartorius muscles of the frog have been investigated. Cation conductance was measured under current clamp conditions using a double sucrosegap technique. Cation effluxes were studied by means of flame emission photometry. All three antibiotics increased the cation conductance and efflux rates; however, differences between the polyenes were found in the steady state values of induced cation transport at a given concentration. The values of both induced conductance g_A and efflux rate constants K_A formed the following sequence: levorin > mycoheptin > nystatin, demonstrating a correlation with the order of antifungal activities. The dose-response curves of lg polyene-induced cation transport against lg of antibiotic concentration in our experiments had slope values which were much lower than those in bilayers: 1.7 and 1.3 for nystatin and mycoheptin, respectively, whereas the aromatic heptaene levorin had an even smaller concentration dependence. The decline in the equilibrium conductance caused by nystatin- and mycoheptin removal was very fast (during the first minute = 0.74 and 2.39 min, respectively). In contrast, levorin-induced conductance was irreversible. It is proposed that the processes which limit the rate of channel formation are different in biological and model membranes. Correspondence to: N. E. Shvinka     

Extracellular calcium and the inotropic effect of epinephrine on frog skeletal muscle

The purpose of these experiments was to determine if extracellular calcium plays an important role in mediating the inotropic effect of epinephrine in isolated frog sartorius muscle. Initial experiments indicated that epinephrine potentiated the muscle twitch in a concentration-dependent manner with concentrations of 10 microM to 1 mM, increasing peak tension by approximately 33%. To inhibit the influx of extracellular calcium, muscles were incubated for 20 min in media containing epinephrine in which calcium had been removed and replaced by magnesium or EDTA, or in experimental media containing epinephrine and the calcium channel blockers D-600 or diltiazem (5 microM). Each experimental condition was found to antagonize the effects of epinephrine such that peak twitch tensions were not significantly different from the control. When muscles were returned to normal Ringer's solution containing epinephrine, twitches exhibited progressive potentiation. Muscles were also incubated for 20 min in epinephrine without stimulation. Once stimulation was resumed, twitches were not immediately potentiated but rather gradually increased over time. These results suggest that the inotropic effects of epinephrine are influenced by the influx of extracellular calcium, an event that is dependent on muscle activation.     

Calcium currents of frog and insect skeletal muscle fibres measured during voltage clamp

Both vertebrate and invertebrate skeletal muscle fibres have Ca2+ permeability mechanisms which are turned on by depolarization of the surface membrane. In frog muscle, Ca currents are extremely slow and will be scarcely activated during the action potential that normally elicits a twitch. This Ca permeability cannot therefore play any substantial, direct role in excitation--contraction coupling. In insect (Carausius morosus) muscle, Ca currents activate within milliseconds of depolarization, even at low temperature, and may well play at least a triggering role in excitation--contraction coupling. These Ca currents show saturation with increasing [Ca]0, while the instantaneous current--voltage relation rectifies inwards, as expected from a very low [Ca]i. The Ca channel is permeable to Sr2+ and Ba2+. Inactivation of Ca currents under a maintained depolarization depends on Ca2+ carrying inward current, however, rather than on the depolarization itself.     

Rapid relaxation of single frog skeletal muscle fibres following laser flash photolysis of the caged calcium chelator, diazo-2

Diazo-2 is a calcium chelator based on BAPTA [(1989) J. Biol. Chem., in press], whose electron withdrawing diazoacetyl group may be rapidly (2000 s-1) converted photochemically to an electron donating carboxymethyl group by exposure to near ultraviolet light, producing an increase in its calcium affinity (Kd changes from 2.2 microM to 0.073 microM) without steric modification of the metal binding site. Photolysis of a 2 mM solution of this compound with a brief flash of light from a frequency-doubled ruby laser (347 nm) caused single skinned muscle fibres from the semitendinosus muscle of the frog Rana temporaria to relax with a mean half-time of 60.4 +/- 5 ms (range 30-100 ms, n = 15) at 12 degrees C, which is faster than the relaxation observed in intact muscles (half-time 133 ms at 14 degrees C [(1986) J. Mol. Biol. 188, 325-342]) and similar to the rate of the fast phase of tension decay in intact single fibres (20 s-1 at 10 degrees C [(1982) J. Physiol. 329, 1-20]).     

Caffeine slows turn-off of calcium release in voltage clamped skeletal muscle fibers.

Myoplasmic free calcium transients delta [Ca2+] were monitored with the calcium indicators antipyrylazo III and fura-2 in voltage clamped cut frog skeletal muscle fibers, in the presence and absence of 0.5 mM caffeine. Without caffeine delta [Ca2+] began to decline within a few milliseconds of fiber repolarization for pulses of all durations. In caffeine delta [Ca2+] continued to rise for 10-60 ms after 10 or 20 ms depolarizing pulses, indicating that the release of calcium from the sarcoplasmic reticulum (SR) continued well after repolarization of transverse tubular (TT) membranes in the presence of caffeine. Caffeine also increased the peak amplitude of delta [Ca2+] for all pulses and slowed the decline of delta [Ca2+] after pulses of all durations. The rate of calcium release from the SR calculated from delta [Ca2+] showed that for 10 ms pulses in caffeine release did not turn off abruptly on repolarization but instead declined to zero with a time constant essentially the same as the time constant for inactivation of SR calcium release during depolarizing pulses in the presence or absence of caffeine. The observed loss of TT membrane potential control of SR calcium release in the presence of caffeine suggests the appearance of a significant component of cytosolic Ca2+-induced calcium release in caffeine.     

Luminal calcium regulates calcium release in triads isolated from frog and rabbit skeletal muscle.

Triads isolated from frog and rabbit skeletal muscle were equilibrated with different external [Ca2+], ranging from 0.025 to 10 mM. Vesicular calcium increased with external [Ca2+] as the sum of a linear plus a saturable component; the latter, which vanished after calsequestrin removal, displayed Bmax values of 182 and 132 nmol of calcium/mg of protein, with Kd values of 1.21 and 1.14 mM in frog and rabbit vesicles, respectively. The effect of luminal [Ca2+] on release kinetics in triads from frog and rabbit skeletal muscle was investigated, triggering release with 2 mM ATP, pCa 5, pH 6.8. In triads from frog, release rate constant (k) values increased sixfold after increasing luminal [Ca2+] from 0.025 to 3 mM. In triads from rabbit, k values increased 20-fold when luminal [Ca2+] increased from 0.05 to 0.7 mM. In both preparations, k values remained relatively constant (10-12 s-1) at higher luminal [Ca2+], with a small decrease at 10 mM. Initial release rates increased with luminal [Ca2+] in both preparations; in triads from rabbit the increase was hyperbolic, and in triads from frogs the increase was sigmoidal. These results indicate that, although triads from frog and rabbit respond differently, in both preparations luminal [Ca2+] has a distinctive effect on release, presumably by regulating sarcoplasmic reticulum calcium channels.