Calcium signals recorded from cut frog twitch fibers containing tetramethylmurexide

The Ca indicator tetramethylmurexide was introduced into cut fibers, mounted in a double-Vaseline-gap chamber, by diffusion from the end-pool solutions. The indicator diffused rapidly to the central region of a fiber where optical recording was done and, if removed, diffused away equally fast. The time course of concentration suggests that, on average, a fraction 0.27 of indicator was reversibly bound to myoplasmic constituents and the free diffusion constant was 1.75 x 10(-6) cm2/s at 18 degrees C. The shape of the resting absorbance spectrum suggests that a fraction 0.11-0.15 of tetramethylmurexide inside a fiber was complexed with Ca. After action potential stimulation, there was a rapid transient change in indicator absorbance followed by a maintained change of opposite sign. The wavelength dependence of both changes matched a cuvette Ca-difference spectrum. The amplitude of the early peak varied linearly with indicator concentration and corresponded to an average rise in free [Ca] of 17 microM. These rather diverse findings can be explained if the sarcoplasmic reticulum membranes are permeable to Ca-free indicator. Both Ca-free and Ca-complexed indicator inside the sarcoplasmic reticulum would appear to be bound by diffusion analysis and the Ca-complexed form would be detected by the resting absorbance spectrum. The transient change in indicator absorbance would be produced by myoplasmic Ca reacting with indicator molecules that freely diffuse in myoplasmic solution. The maintained signal, which reports Ca dissociating from indicator complexed at rest, would come from changes within the sarcoplasmic reticulum. A method, based on these ideas, is described for separating the two components of the tetramethylmurexide signal. The estimated myoplasmic free [Ca] transient has an average peak value of 26 microM at 18 degrees C. Its time course is similar to, but possibly faster than, that recorded with antipyrylazo III (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:83-143).     

Calcium signals recorded from cut frog twitch fibers containing antipyrylazo III

The Ca indicator antipyrylazo III was introduced into cut frog twitch fibers by diffusion (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:41-81). Like arsenazo III, antipyrylazo III was largely bound to or sequestered by intracellular constituents; on average, a fraction 0.68 was so immobilized. After action potential stimulation, there was an early change in absorbance, with a wavelength dependence that nearly matched a cuvette Ca-difference spectrum. As with arsenazo III, this signal became prolonged as experiments progressed. In a freshly prepared cut fiber containing 0.3 mM indicator, the absorbance change had an average half-width of 10 ms at 18 degrees C. The peak amplitude of this Ca signal depended on the indicator concentration in a roughly parabolic manner, which is consistent with a 1:2 stoichiometry for Ca:indicator complexation and, for indicator concentrations less than or equal to 0.4 mM, constant peak free [Ca]. If all the antipyrylazo III inside a fiber can react normally with Ca, peak free [Ca] is 3 microM at 18 degrees C. If only freely diffusible indicator can react, the estimate is 42 microM. The true amplitude probably lies somewhere in between. The time course of Ca binding to intracellular buffers and of Ca release from the sarcoplasmic reticulum is estimated from the 3- and 42-microM myoplasmic [Ca] transients. After action potential stimulation, the release waveform is rapid and brief; its latency after the surface action potential is 2-3 ms and its half-width is 2-4 ms. This requires rapid coupling between the action potential in the transverse tubular system and Ca release from the sarcoplasmic reticulum. The peak fractional occupancy calculated for Ca-regulatory sites on troponin is 0.46 for the 3-microM transient and 0.93 for the 42-microM transient. During a 100-ms tetanus at 100 Hz, the corresponding fractional occupancies are 0.56 and 0.94. The low value of occupancy associated with the low-amplitude [Ca] calibration seems inconsistent with a brief tetanus being able to produce near-maximal activation (Blinks, J. R., R. Rudel, and S. R. Taylor. 1978. Journal of Physiology. 277:291-323; Lopez J. R., L. A. Wanck, and S. R. Taylor. 1981. Science. 214:47-82).     

Comparison of arsenazo III optical signals in intact and cut frog twitch fibers

The Ca indicator arsenazo III was introduced into cut frog twitch fibers by diffusion from end-pool segments rendered permeable by saponin. After 2-3 h, the arsenazo III concentration at the optical recording site in the center of a fiber reached two to three times that in the end-pool solutions. Thus, arsenazo III was bound to or taken up by intracellular constituents. The time course of indicator appearance was fitted by equations for diffusion plus linear reversible binding; on average, 0.73 of the indicator was bound and the free diffusion constant was 0.86 x 10(-6) cm2/s at 18 degrees C. When the indicator was removed from the end pools, it failed to diffuse away from the optical site as rapidly as it had diffused in. The wavelength dependence of resting arsenazo III absorbance was the same in cut fibers and injected intact fibers. After action potential stimulation, the active Ca and dichroic signals were similar in the two preparations, which indicates that arsenazo III undergoes the same changes in absorbance and orientation in both cut and intact fibers. Ca transients in freshly prepared cut fibers appeared to be similar to those in intact fibers. As a cut fiber experiment progressed, however, the Ca signal changed. With action potential stimulation, the half-width of the signal gradually increased, regardless of whether the indicator concentration was increasing or decreasing. This increase was usually not accompanied by any change in the amplitude of the Ca signal at a given indicator concentration or by any obvious deterioration in the electrical condition of the fiber. In voltage-clamp experiments near threshold, the relation between peak [Ca] and voltage usually became less steep with time and shifted to more negative potentials. All these changes were also observed in cut fibers containing antipyrylazo III (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:83-143). They are considered to represent a progressive change in the physiological state of a cut fiber during the time course of an experiment.     

Calcium release in frog cut twitch fibers exposed to different ionic environments under voltage clamp.

Calcium release was measured in highly stretched frog cut twitch fibers mounted in a double Vaseline-gap voltage clamp chamber, with the internal solution containing 20 mM EGTA plus 0.4 or 1.8 mM added calcium. Rise in myoplasmic [Ca(2+)] was monitored with antipyrylazo III as the indicator at a temperature of 13 to 14 degrees C. The waveform of calcium release rate (Rel) computed from the absorbance change showed an early peak (Rel(p)) followed by a maintained phase (Rel(m)). Each Rel(p)-versus-V plot was fitted with a Boltzmann distribution function. The maximum value of Rel(p) (Rel(p,max)) was compared in various calcium-containing external solutions. The average value in a Cl(-) solution was about one-third larger than those in a CH(3)SO(3)(-) or gluconate solution, whereas the values in the CH(3)SO(3)(-) and gluconate solutions had no statistically significant difference. In external solutions containing CH(3)SO(3)(-) or gluconate, a replacement of the Ca(2+) with Mg(2+) reduced Rel(p,max) by 30 to 50%, on average. The values of Rel(p, max) also had no statistically significant difference among calcium-free external solutions containing different impermeant anions. An increase of the nominal free [Ca(2+)] in the end-pool solution from a reduced to the normal physiological level increased the value of Rel(p,max), and also slowed the decay of the maintained phase of the Rel waveform. The Rel waveforms in the Cl(-) and CH(3)SO(3)(-) solutions were compared in the same fiber at a fixed potential. CH(3)SO(3)(-) increased the time to peak, reduced Rel(p), and increased Rel(m), and the effects were partially reversible. Under the hypothesis that the decay of the peak was due to calcium inactivation of calcium release, the inactivation was larger in Cl(-) than in CH(3)SO(3)(-), in qualitative agreement with the ratio of Rel(p) in the two solutions. Under the alternative hypothesis that the peak and the maintained phase were separately gated by calcium and depolarization, respectively, then CH(3)SO(3)(-) appeared to decrease the calcium-gated component and increase the voltage-gated component.     

Intrinsic optical and passive electrical properties of cut frog twitch fibers

This article describes a new apparatus for making simultaneous optical measurements on single muscle fibers at three different wavelengths and two planes of linear polarization. There are two modes of operation: mode 1 measures the individual absorbances of light linearly polarized along and perpendicular to the fiber axis, and mode 2 measures retardation (or birefringence) and the average of the two absorbance components. Although some intact frog twitch fibers were studied, most experiments used cut fibers (Hille, B., and D. T. Campbell. 1976. Journal of General Physiology. 67:265-293) mounted in a double-Vaseline-gap chamber (Kovacs, L., E. Rios, and M. F. Schneider. 1983. Journal of Physiology. 343:161-196). The end-pool segments were usually exposed for 2 min to 0.01% saponin. This procedure, used in subsequent experiments to make the external membranes in the end pools permeable to Ca indicators (Maylie, J., M. Irving, N. L. Sizto, G. Boyarsky, and W. K. Chandler. 1987. Journal of General Physiology. 89:145-176; Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:41-143), was routinely employed so that all our cut fiber results would be comparable. A simple method, which does not require microelectrodes, allowed continual estimation of a fiber's membrane (rm) and internal longitudinal (ri) resistances as well as the external resistance (re) under the Vaseline seals. The values of rm and ri obtained from cut fibers with this method agree reasonably well with values obtained from intact fibers using microelectrode techniques. Optical measurements were made on resting and action potential-stimulated fibers. The intrinsic fiber absorbance, defined operationally as log10 of the ratio of incident light to transmitted light intensity, was similar in intact and cut preparations, as were the changes that accompanied stimulation. On the other hand, the resting birefringence and the peak of the active change in cut fibers were, respectively, only 0.8 and 0.7 times the corresponding values in intact fibers. Both the amplitude and the half-width of the active retardation signal increased considerably during the time course of cut fiber experiments; a twofold increase in 2 h was not unusual. Such changes are probably due to a progressive alteration in the internal state of the cut fibers.     

Calcium influx in contracting and paralyzed frog twitch muscle fibers

Calcium uptake produced by a potassium contracture in isolated frog twitch fibers was 6.7 +/- 0.8 pmol in 0.7 cm of fiber (mean +/- SEM, 21 observations) in the presence of 30 microM D600. When potassium was applied to fibers paralyzed by the combination of 30 microM D600, cold, and a prior contracture, the calcium uptake fell to 3.0 +/- 0.7 pmol (11): the fibers were soaked in 45Ca in sodium Ringer for 3 min before 45Ca, in a potassium solution, was added for 2 min; each estimate of uptake was corrected for 5 min of resting influx, measured from the same fiber (average = 2.3 +/- 0.3 pmol). The calcium influx into paralyzed fibers is unrelated to contraction. This voltage-sensitive, slowly inactivating influx, which can be blocked by 4 mM nickel, has properties similar to the calcium current described by several laboratories. The paired difference in calcium uptake between contracting and paralyzed fibers, 2.9 +/- 0.8 pmol (16), is a component of influx related to contraction. Its size varies with contracture size and it occurs after tension production: 45Ca applied immediately after contracture is taken up in essentially the same amounts as 45Ca added before contraction. This delayed uptake is probably a "reflux" refilling a binding site on the cytoplasmic side of the T membrane, which had been emptied during the prior contracture, perhaps to initiate it. We detect no component of calcium uptake related to excitation-contraction coupling occurring before or during a contracture.     

Effect of fura-2 on action potential-stimulated calcium release in cut twitch fibers from frog muscle

Cut fibers (striation spacing, 3.6-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14-15 degrees C. One or both of the Ca indicators fura-2 and purpurate-3,3' diacetic acid (PDAA) were introduced into the optical recording site by diffusion from the end pools. Sarcoplasmic reticulum (SR) Ca release was elicited by action potential stimulation. With resting [fura-2] = 0 mM at the optical site, the [Ca] transient measured with PDAA was used to estimate SR Ca release (Baylor, S.M., W.K. Chandler, and M.W. Marshall. 1983. Journal of Physiology. 344:625-666). With resting [fura-2] > 0 mM, the contribution from Ca complexation by fura-2 was added to the estimate. When resting [fura-2] was increased from 0 to 0.5-2 mM, both the amount of SR Ca release and the maximal rate of release were increased by approximately 20%. These results are qualitatively similar to those obtained in intact fibers (Baylor, S.M., and S. Hollingworth. 1988. Journal of Physiology. 403:151-192; Hollingworth, S., A. B. Harkins, N. Kurebayashi, M. Konishi, and S. M. Baylor. 1992. Biophysical Journal. 63:224-234) and are consistent with a reduction of Ca inactivation of SR Ca release produced by 0.5-2 mM fura-2. With resting [fura-2] > or = 2 mM, the PDAA [Ca] transient was reduced to nearly zero and SR Ca release could be estimated from delta [Cafura-2] alone. When resting [fura-2] was increased from 2-4 to 5-6 mM, both the amount of SR Ca release and the maximal rate of release were decreased by approximately half, consistent with a possible reduction of Ca- induced Ca release (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophysical Journal. 60:867-873) or a possible pharmacological effect of fura-2.     

Membrane capacitance in frog cut twitch fibers mounted in a double vaseline-gap chamber

In experiments on cut muscle fibers mounted in a double Vaseline-gap chamber, electrical measurements are usually made by measuring the voltage V1(t) in one end pool and by passing current I2(t) from the other end pool to the central pool, which is usually clamped to earth potential. The voltage in the current-passing end pool is denoted by V2(t). This article describes how the value of the holding current, Ih, and the values of delta V2(infinity)/delta V1(infinity) and delta I2(infinity)/delta V1(infinity) that are associated with a small change in V1(t) can be used to estimate the linear cable parameters rm, ri, and re in a cut fiber that has been equilibrated with a Cs-containing internal solution. rm, ri, and re represent, respectively, the resistance of the plasma membranes, the internal longitudinal resistance, and the external longitudinal resistance under the Vaseline seals, all for a unit length of fiber. The apparent capacitance, Capp, of the preparation is defined to equal integral of infinity 0 delta I2,tr(t) dt/delta V1(infinity), in which delta I2,tr(t) represents the transient component of current that is associated with a change in V1(t) of amplitude delta V1(infinity). A method is described to estimate cm, the capacitance of the plasma membranes per unit length of fiber, from Capp and the values of rm, ri, and re. In experiments carried out with a tetraethylammonium chloride (TEA.Cl) solution at 13-14 degrees C in the central pool, cm remained stable for as long as 3-4 h. The values of cm, 0.19 microF/cm on average, and their variation with fiber diameter are similar to published results from intact fibers. This article also describes the different pathways that are taken by the current that flows from the current-passing end pool to the central pool. Approximately two-thirds of delta I2,tr(t) flows across the capacitance of the plasma membranes in the central-pool region. The rest flows either across plasma membranes that are under the two Vaseline seals or directly from the current-passing end pool to the central pool, across the external longitudinal resistance under the Vaseline seal. [There is also a current that flows directly from the voltage-measuring end pool to the central pool but this does not contribute to delta I2,tr(t).]     

Existence of Q gamma in frog cut twitch fibers with little Q beta.

Charge movements were measured in frog cut twitch fibers with the double Vaseline-gap voltage-clamp technique. In most fibers, when a depolarizing pulse to -60 to -40 mV was applied at 13-14 degrees C, the ON segment of a charge movement trace showed an early I beta component and a late I gamma hump component. An ongoing controversy is whether the I gamma hump component triggers calcium release from the sarcoplasmic reticulum or arises as a consequence of the release. Interestingly, a number of cut fibers showed normal I gamma components but greatly diminished, or unresolvable, I beta components. When the amount of charge associated with the current transient was plotted against the membrane potential, the steeply voltage-dependent Q gamma component appeared normal whereas the less steeply voltage-dependent Q beta component was also greatly diminished or unresolvable. These results suggest that I gamma can flow in the absence of I beta, thereby ruling out the possibility that Q beta triggers calcium release which, in turn, causes Q gamma to move. The results, however, do not rule out the positive feedback of calcium release to activate Q gamma, if calcium release is not triggered by Q beta but by Q gamma itself or by some other signal.     

Intramembranous charge movement in frog cut twitch fibers mounted in a double vaseline-gap chamber

Intramembranous charge movement was measured in cut twitch fibers mounted in a double Vaseline-gap chamber with either a tetraethylammonium chloride (TEA.Cl) or a TEA2.SO4 solution (13-14 degrees C) in the central pool. Charge vs. voltage data were fitted by a single two-state Boltzmann distribution function. The average values of V (the voltage at which steady-state charge is equally distributed between the two Boltzmann states), k (the voltage dependence factor), and qmax/cm (the maximum charge divided by the linear capacitance, both per unit length of fiber) were V = -53.3 mV (SEM, 1.1 mV), k = 6.3 mV (SEM, 0.3 mV), qmax/cm = 18.0 nC/microF (SEM, 1.1 nC/microF) in the TEA.Cl solution; and V = -35.1 mV (SEM, 1.8 mV), k = 10.5 mV (SEM, 0.9 mV), qmax/cm = 36.3 nC/microF (SEM, 3.2 nC/microF) in the TEA2.SO4 solution. These values of k are smaller than those previously reported for cut twitch fibers and are as small as those reported for intact fibers. If a correction is made for the contributions of currents from under the Vaseline seals, V = -51.2 mV (SEM, 1.1 mV), k = 7.2 mV (SEM, 0.4 mV), qmax/cm = 22.9 nC/microF (SEM, 1.4 nC/microF) in the TEA.Cl solution; and V = -34.0 mV (SEM, 1.9 mV), k = 10.1 mV (SEM, 1.1 mV), qmax/cm = 38.8 nC/microF (SEM, 3.2 nC/microF) in the TEA2.SO4 solution. With this correction, however, the fit of the theoretical curve to the data is poor. A good fit with this correction can be obtained with a sum of two Boltzmann distribution functions. The first has average values V = -33.0 mV (SEM, 2.8 mV), k = 11.0 mV (SEM, 0.5 mV), qmax/cm = 10.6 nC/microF (SEM, 1.0 nC/microF) in the TEA.Cl solution; and V = -20.0 mV (SEM, 3.3 mV), k = 17.0 mV (SEM, 2.0 mV), qmax/cm = 36.4 nC/microF (SEM, 2.3 nC/microF) in the TEA2.SO4 solution. The second has average values V = -56.5 mV (SEM, 1.3 mV), k = 2.9 mV (SEM, 0.4 mV), qmax/cm = 13.2 nC/microF (SEM, 1.0 nC/microF) in the TEA.Cl solution; and V = -41.6 mV (SEM, 1.4 mV), k = 2.5 mV (SEM, 0.8 mV), qmax/cm = 11.8 nC/microF (SEM, 1.7 nC/microF) in the TEA2.SO4 solution. When a fiber is depolarized to near V of the second Boltzmann function, a slowly developing "hump" appears in the ON-segment of the current record.(ABSTRACT TRUNCATED AT 400 WORDS)     

Q beta and Q gamma components of intramembranous charge movement in frog cut twitch fibers

Intramembranous charge movement was measured in frog cut twitch fibers mounted in a double Vaseline-gap chamber with a TEA.Cl solution at 13-14 degrees C in the central pool. When a fiber was depolarized from a holding potential of -90 mV to a potential near -60 mV, the current from intramembranous charge movement was outward in direction and had an early, rapid component and a late, more slowly developing component, referred to as I beta and I gamma, respectively (1979. J. Physiol. [Lond.]. 289:83-97). When the pulse to -60 mV was preceded by a 100-600-ms pulse to -40 mV, early I beta and late I gamma components were also observed, but in the inward direction. The shape of the Q gamma vs. voltage curve can be estimated with this two-pulse protocol. The first pulse to voltage V allows the amounts of Q beta and Q gamma charge in the active state to change from their respective resting levels, Q beta (-90) and Q gamma (-90), to new steady levels, Q beta (V) and Q gamma (V). A second 100-120-ms pulse, usually to -60 mV, allows the amount of Q beta charge in the active state to change from Q beta (V) to Q beta (-60) but is not sufficiently long for the amount of Q gamma charge to change completely from Q gamma (V) to Q gamma (-60). The difference between the amount of Q gamma charge at the end of the second pulse and Q gamma (-60) is estimated from the OFF charge that is observed on repolarization to -90 mV. The OFF charge vs. voltage data were fitted, with gap corrections, with a Boltzmann distribution function plus a constant. The mean values of V (the potential at which, in the steady state, charge is distributed equally between the resting and active states) and k (the voltage dependence factor) were -59.2 mV (SEM, 1.1 mV) and 1.2 mV (SEM, 0.6 mV), respectively. The one-pulse charge vs. voltage data from the same fibers were fitted with a sum of two Boltzmann functions (1990. J. Gen. Physiol. 96:257-297). The mean values of V and k for the steeply voltage-dependent Boltzmann function, which is likely to be associated with the Q gamma component of charge, were -55.3 mV (SEM, 1.3 mV) and 3.3 mV (SEM, 0.6 mV), respectively, similar to the corresponding values obtained with the two-pulse protocol.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium efflux from frog twich muscle fibers

An apparatus is described which collects the effluent from the center 0.7 cm of a single muscle fiber or bundle of muscle fibers. It was used to study the efflux of ⁴⁵Ca from twitch muscle fibers. The efflux can be described by three time constants 18 ± 2 min, 300 ± 40 min, and 882 ± 172 min. These kinetics have been interpreted as those of a three-compartment system. The fastest is thought to be on the surface membrane of the muscle and of the T system. It contains 0.07 ± 0.03 mM Ca/liter of fiber and the Ca efflux is 0.11 ± 0.04 pM Ca/cm². sec. The intermediate rate compartment is thought to represent the Ca in the longitudinal reticulum. It contains approximately 0.77 mM Ca/liter. Only the efflux from this compartment increases during stimulation. The most slowly exchanging compartment is poorly defined. Neither Ca-free nor Ni-Ringer solutions alter the rate of loss from the fastest exchanging compartment. Ni apparently alters the rate of loss from the slowest compartment.     

Factors affecting the appearance of the hump charge movement component in frog cut twitch fibers.

Charge movement was measured in frog cut twitch fibers with the double Vaseline gap technique. Five manipulations listed below were applied to investigate their effects on the hump component (I gamma) in the ON segments of TEST minus CONTROL current traces. When external Cl-1 was replaced by MeSO3- to eliminate Cl current, I gamma peaked earlier due to a few millivolts shift of the voltage dependence of I gamma kinetics in the negative direction. The Q-V plots in the TEA.Cl and TEA.MeSO3 solutions were well fitted by a sum of two Boltzmann distribution functions. The more steeply voltage-dependent component (Q gamma) had a V approximately 6 mV more negative in the TEA.MeSO3 solution than in the TEA.Cl solution. These voltage shifts were partially reversible. When creatine phosphate in the end pool solution was removed, the I gamma hump disappeared slowly over the course of 20-30 min, partly due to a suppression of Q gamma. The hump reappeared when creatine phosphate was restored. When 0.2-1.0 mM Cd2+ was added to the center pool solution to block inward Ca current, the I gamma hump became less prominent due to a prolongation in the time course of I gamma but not to a suppression of Q gamma. When the holding potential was changed from -90 to -120 mV, the amplitude of I beta was increased, thereby obscuring the I gamma hump. Finally, when a cut fiber was stimulated repetitively, I gamma lost its hump appearance because its time course was prolonged. In an extreme case, a 5-min resting interval was insufficient for a complete recovery of the waveform. In general, a stimulation rate of once per minute had a negligible effect on the shape of I gamma. Of the five manipulations, MeSO3- has the least perturbation on the appearance of I gamma and is potentially a better substitute for Cl- than SO2-(4) in eliminating Cl current if the appearance of the I gamma hump is to be preserved.     

Reduction of calcium inactivation of sarcoplasmic reticulum calcium release by fura-2 in voltage-clamped cut twitch fibers from frog muscle

Cut fibers from Rana temporaria and Rana pipiens (striation spacing, 3.9-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14 degrees C. The Ca indicator purpurate-3,3' diacetic acid (PDAA) was introduced into the end pools and allowed to diffuse into the optical recording site. When the concentration at the site exceeded 2 mM, step depolarizations to 10 mV were applied and the [Ca] transient measured with PDAA was used to estimate Ca release from the sarcoplasmic reticulum (SR) (Baylor, S. M., W. K. Chandler, and M. W. Marshall. 1983. Journal of Physiology. 344:625-666). With depolarization, the rate of SR Ca release increased to an early peak and then rapidly decreased several-fold to a quasi-steady level. The total amount of Ca released from the SR at the time of peak rate of release appeared to be independent of SR Ca content, consistent with the idea that a single activated channel might pass, on average, a fixed number of ions, independent of the magnitude of the single channel flux. A possible explanation of this property is given in terms of locally induced Ca inactivation of Ca release. The solution in the end pools was then changed to one with PDAA plus fura-2. SR Ca release was estimated from the [Ca] transient, as before, and from the delta [Cafura-2] signal. On average, 2-3 mM fura-2 increased the quasi-steady level of the rate of SR Ca release by factors of 6.6 and 3.8, respectively, in three fibers from Rana temporaria and three fibers from Rana pipiens. The peak rate of release was increased in five of the six fibers but to a lesser extent than the quasi-steady level. In all fibers, the amplitude of the free [Ca] transient was markedly reduced. These increases in the rate of SR Ca release are consistent with the idea that Ca inactivation of Ca release develops during a step depolarization to 10 mV and that 2-3 mM fura-2 is able to reduce this inactivation by complexing Ca and thereby reducing free [Ca]. Once the concentration of fura-2 becomes sufficiently large, a further increase reduces the rate of SR Ca release. On average, 5-6 mM fura-2 increased the quasi-steady rate of release, compared with 0 mM fura-2, by 6.5 and 2.9, respectively, in four fibers from Rana temporaria and three from Rana pipiens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Simultaneous monitoring of changes in magnesium and calcium concentrations in frog cut twitch fibers containing antipyrylazo III

Antipyrylazo III was introduced into frog cut twitch fibers (17-19 degrees C) by diffusion. After action potential stimulation, the change in indicator absorbance could be resolved into two components that had different time courses and wavelength dependences. The first component was early and transient and due to an increase in myoplasmic free [Ca] (Maylie, J., M. Irving, N.L. Sizto, and W.K. Chandler, 1987, Journal of General Physiology, 89:83-143). The second component, usually measured at 590 nm (near the isosbestic wavelength for Ca), developed later than the Ca transient and returned towards baseline about 100 times more slowly. Although the wavelength dependence of this component is consistent with an increase in either free [Mg] or pH, its time course is clearly different from that of the signals obtained with the pH indicators phenol red and 4',5'-dimethyl-5-(and -6-) carboxyfluorescein, suggesting that it is mainly due to an increase in free [Mg]. After a single action potential in freshly prepared cut fibers that contained 0.3 mM antipyrylazo III, the mean peak amplitude of delta A (590) would correspond to an increase in free [Mg] of 47 microM if all the signal were due to a change in [Mg] and all the intracellular indicator reacted with Mg as in cuvette calibrations. With either repetitive action potential stimulation or voltage-clamp depolarization, the delta A (590) signal continued to develop throughout the period when free [Ca] was elevated and then recovered to within 40-90% of the prestimulus baseline with an average rate constant between 0.5 and 1.0 s-1. With prolonged voltage-clamp depolarization, both the amplitude and rate of development of the delta A(590) signal increased with the amplitude of the depolarization and appeared to saturate at levels corresponding to an increase in free [Mg] of 0.8-1.4 mM and a maximum rate constant of 3-4 s-1, respectively. These results are consistent with the idea that the delta A(590) signal is primarily due to changes in myoplasmic free [Mg] produced by a change in the Mg occupancy of the Ca,Mg sites on parvalbumin that results from the Ca transient.     

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)     

Comparison of charge movement components in intact and cut twitch fibers of the frog. Effects of stretch and temperature

Charge movements were measured in frog intact fibers with the three-microelectrode technique and in cut fibers with the double Vaseline gap technique. At 13-14 degrees C, the ON segments of charge movement records from both preparations showed an early I beta component and a late I gamma hump component. When an intact fiber was cooled to 4-7 degrees C, the time-to-peak of I gamma (tp,gamma) was prolonged, but I gamma still appeared as a hump. Q-V plots from intact fibers at 4-7 degrees C were fitted with a sum of two Boltzmann distribution functions (method 1). The more steeply voltage-dependent component, identified with Q gamma, accounted for 32.1% (SEM 2.2%) of the total charge. This fraction was larger than the 22.6% (SEM 1.5%) obtained by separating the ON currents with a sum of two kinetic functions (method 2). The total charge in cut fibers stretched to a sarcomere length of 3.5 microns at 13-14 degrees C was separated into Q beta and Q gamma by methods 1 and 2. The fraction of Q gamma in the total charge was 51.3% (SEM 1.7%) and 53.7% (SEM 1.8%), respectively, suggesting that cut fibers have a larger proportion of Q gamma:Q beta than intact fibers. When cut fibers were stretched to a sarcomere length of 4 microns, the proportion of Q gamma:Q beta was unchanged. Between 4 and 13 degrees C, the Q10 of l/tp,gamma in intact fibers was 2.33 (SEM 0.33) and that of 1/tau beta was less than 1.44 (SEM 0.04), implying that the kinetics of I gamma has a steeper temperature dependence than the kinetics of I beta. When cut fibers were cooled from 14 to 6 degrees C, I gamma in the ON segment generally became too broad to be manifested as a hump. In a cut fiber in which I gamma was manifested as a hump, the Q10 of l/tp,gamma was 2.08 and that of l/tau beta was less than 1.47. Separating the Q-V plots from cut fibers at different temperatures by method 1 showed that the proportion of Q gamma:Q beta was unaffected by temperature change. The appearance of I gamma humps at low temperatures in intact fibers but generally not in cut fibers suggests an intrinsic difference between the two fiber preparations.     

Effects of conditioning depolarization and repetitive stimulation on Q beta and Q gamma charge components in frog cut twitch fibers

Charge movement was measured in frog cut twitch fibers with the double Vaseline-gap technique. Steady-state inactivation of charge movement was studied by changing the holding potential from -90 mV to a level ranging from -70 to -30 mV. Q beta and Q gamma at each holding potential were separated by fitting the Q-V plot with a sum of two Boltzmann distribution functions. At -70 mV Q beta and Q gamma were inactivated to 54.0% (SEM 2.2) and 82.7% (SEM 3.0) of the amounts at -90 mV. At holding potentials greater than or equal to -60 mV, more Q gamma was inactivated than Q beta, and at -30 mV Q gamma was completely inactivated but Q beta was not. There was no holding potential at which Q beta was unaffected and Q gamma was completely inactivated. The differences between the residual fractions of Q beta and Q gamma are significant at all holding potentials (P less than 0.001-0.05). The plot of the residual fraction of Q beta or Q gamma versus holding potential can be fitted well by an inverted sigmoidal curve that is a mirror image of the activation curve of the respective charge component. The pair of curves for Q gamma correlates well with those for tension generation or Ca release obtained by other investigators. The time courses of the inactivation of Q beta and Q gamma were studied by obtaining several Q-V plots with conditioning depolarizations lasting 1-20 s and separating each Q-V plot into Q beta and Q gamma components by fitting with a sum of two Boltzmann distribution functions. The inactivation time constant of Q beta was found to be 5-10 times as large as that of Q gamma. During repetitive stimulation, prominent I gamma humps could be observed in TEST-minus-CONTROL current traces and normal Q gamma components could be separated from the Q-V plots, whether 20 or 50 mM EGTA was present in the internal solution, whether 2 or 10 stimulations were used, and whether the stimuli were separated by 400 ms or 6 s. Repetitive stimulation slowed the kinetics of the I gamma hump and could shift the Q-V curve slightly in the depolarizing direction in some cases, resulting in an apparent suppression of charge at the potentials that fall on the steep part of the Q-V curve.     

Calcium release and its voltage dependence in frog cut muscle fibers equilibrated with 20 mM EGTA

Sarcoplasmic reticulum (SR) Ca release was studied at 13-16 degrees C in cut fibers (sarcomere length, 3.4-3.9 microns) mounted in a double Vaseline-gap chamber. The amplitude and duration of the action- potential stimulated free [Ca] transient were reduced by equilibration with end-pool solutions that contained 20 mM EGTA with 1.76 mM Ca and 0.63 mM phenol red, a maneuver that appeared to markedly reduce the amount of Ca complexed by troponin. A theoretical analysis shows that, under these conditions, the increase in myoplasmic free [Ca] is expected to be restricted to within a few hundred nanometers of the SR Ca release sites and to have a time course that essentially matches that of release. Furthermore, almost all of the Ca that is released from the SR is expected to be rapidly bound by EGTA and exchanged for protons with a 1:2 stoichiometry. Consequently, the time course of SR Ca release can be estimated by scaling the delta pH signal measured with phenol red by -beta/2. The value of beta, the buffering power of myoplasm, was determined in fibers equilibrated with a combination of EGTA, phenol red, and fura-2; its mean value was 22 mM/pH unit. The Ca content of the SR (expressed as myoplasmic concentration) was estimated from the total amount of Ca released by either a train of action potentials or a depleting voltage step; its mean value was 2,685 microM in the action-potential experiments and 2,544 microM in the voltage- clamp experiments. An action potential released, on average, 0.14 of the SR Ca content with a peak rate of release of approximately 5%/ms. A second action potential, elicited 20 ms later, released only 0.6 times as much Ca (expressed as a fraction of the SR content), probably because Ca inactivation of Ca release was produced by the first action potential. During a depolarizing voltage step to 60 mV, the rate of Ca release rapidly increased to a peak value of approximately 3%/ms and then decreased to a quasi-steady level that was only 0.6 times as large; this decrease was also probably due to Ca inactivation of Ca release. SR Ca release was studied with small step depolarizations that open no more than one SR Ca channel in 7,000 and increase the value of spatially averaged myoplasmic free [Ca] by only 0.2 nM.     

Calcium inactivation of calcium release in frog cut muscle fibers that contain millimolar EGTA or Fura-2

Cut muscle fibers from Rana temporaria (sarcomere length, 3.4-4.2 microns) were mounted in a double Vaseline-gap chamber (14-15 degrees C) and equilibrated with end-pool solutions that contained 20 mM EGTA and 1.76 mM Ca. Sarcoplasmic reticulum (SR) Ca release was estimated from changes in pH (Pape, P. C., D.-S. Jong, and W.K. Chandler. 1995. Journal of General Physiology. 106:000-000). Although the amplitude and duration of the [Ca] transient, as well as its spatial spread from the release sites, are reduced by EGTA, SR Ca release elicited by either depolarizing voltage-clamp pulses or action potentials behaved in a manner consistent with Ca inactivation of Ca release. After a step depolarization to -20 or 10 mV, the rate of SR Ca release, corrected for SR Ca depletion, reached a peak value within 5-15 ms and then rapidly decreased to a quasi-steady level that was about half the peak value; the time constant of the last half of the decrease was usually 2- 4 ms. Immediately after an action potential or a 10-15 ms prepulse to - 20 mV, the peak rate of SR Ca release elicited by a second stimulation, as well as the fractional amount of release, were substantially decreased. The rising phase of the rate of release was also reduced, suggesting that at least 0.9 of the ability of the SR to release Ca had been inactivated by the first stimulation. There was little change in intramembranous charge movement, suggesting that the changes in SR Ca release were not caused by changes in its voltage activation. These effects of a first stimulation on the rate of SR Ca release elicited by a second stimulation recovered during repolarization to -90 mV; the time constant of recovery was approximately 25 ms in the action- potential experiments and approximately 50 ms in the voltage-clamp experiments. Fura-2, which is able to bind Ca more rapidly than EGTA and hence reduce the amplitude of the [Ca] transient and its spatial spread from release sites by a greater amount, did not prevent Ca inactivation of Ca release, even at concentrations as large as 6-8 mM. These effects of Ca inactivation of Ca release can be simulated by the three-state, two-step model proposed by Schneider, M. F., and B. J. Simon (1988, Journal of Physiology. 405:727-745), in which SR Ca channels function as a single uniform population of channels. (ABSTRACT TRUNCATED AT 400 WORDS)