Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle

Intact cardiac cells from the adult rat or rabbit ventricle were isolated by enzymatic digestion with a progressive increase of the [free Ca2+] in the solution. These cells were electrically stimulated in the presence of 2.50 mM free Ca2+, and a twitch of maximum amplitude was elicited by the positive inotropic interventions that were found to be optimum. Then the cells were chemically skinned, and the maximum tension induced by a saturating [free Ca2+] was used as a reference to express the tension developed during the twitch of the intact cells. The myoplasmic [free Ca2+] reached during the twitch was inferred from the tension-pCa curve. In mechanically skinned cells of the same animal species, the myoplasmic [free Ca2+] reached during Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum (SR) was inferred by two methods using (a) the tension-pCa curve and (b) a direct calibration of the transients of aequorin bioluminescence. The induction of a maximum Ca2+ release from the SR required a larger Ca2+ preload of the SR and a higher [free Ca2+] trigger in the rabbit than in the rat skinned cells. However, the results obtained with the two methods of inference of the myoplasmic [free Ca2+] suggest that in both animal species a maximum myoplasmic [free Ca2+] of pCa approximately 5.40 was reached during both the optimum Ca2+-induced release of Ca2+ from the SR of the skinned cells and the optimum twitch of the intact cells. This was much lower than the [free Ca2+] necessary for the full activation of the myofilaments (pCa approximately 4.90).     

Rapid ionic modifications during the aequorin-detected calcium transient in a skinned canine cardiac Purkinje cell

A microprocessor-controlled system of microinjections and microaspirations has been developed to change, within approximately 1 ms, the [free Ca2+] at the outer surface of the sarcoplasmic reticulum (SR) wrapped around individual myofibrils (0.3-0.4 micron radius) of a skinned canine cardiac Purkinje cell (2.5-4.5 micron overall radius) at different phases of a Ca2+ transient. Simultaneously monitoring tension and aequorin bioluminescence provided two methods for estimating the peak myoplasmic [free Ca2+] reached during the spontaneous cyclic Ca2+ release from the SR obtained in the continuous presence of a bulk solution [free Ca2+] sufficiently high to overload the SR. These methods gave results in excellent agreement for the spontaneous Ca2+ release under a variety of conditions of pH and [free Mg2+], and of enhancement of Ca2+ release by calmodulin. Disagreement was observed, however, when the Ca2+ transient was modified during its ascending phase. The experiments also permitted quantification of the aequorin binding within the myofibrils and determination of its operational apparent affinity constant for Ca2+ at various [free Mg2+] levels. An increase of [free Ca2+] at the outer surface of the SR during the ascending phase of the Ca2+ transient induced further release of Ca2+. In contrast, an increase of [free Ca2+] during the descending phase of the Ca2+ transient did not cause further Ca2+ release. Varying [free H+], [free Mg2+], or the [Na+]/[K+] ratio had no significant effect on the Ca2+ transient during which the modification was applied, but it altered the subsequent Ca2+ transient. Therefore, Ca2+ appears to be the major, if not the only, ion controlling Ca2+ release from the SR rapidly enough to alter a Ca2+ transient during its course.     

Effects of ryanodine in skinned cardiac cells

Ryanodine (1 X 10(-5) M) did not affect the Ca2+ sensitivity of the myofilaments of skinned (sarcolemma removed by microdissection) cardiac cells from the rat ventricle. Ryanodine (1 X 10(-5) M) inhibited three types of Ca2+ release from the sarcoplasmic reticulum (SR), which have different mechanisms: 1) Ca2+-induced release of Ca2+ triggered by a rapid and transient increase of [free Ca2+] at the outer surface of the SR; 2) caffeine-induced release of Ca2+; 3) spontaneous cyclic release of Ca2+ occurring in the continuous presence of a [free Ca2+] sufficient to overload the SR. These results suggest that the three types of Ca2+ release are through the same channel across the SR membrane, although the gating mechanisms are different for the three types. Ryanodine also diminished the rate of Ca2+ accumulation into the SR. Even in the presence of 1 X 10(-5) M ryanodine the SR accumulated Ca2+ that could be released when the SR was sufficiently overloaded with Ca2+. Thus, ryanodine pretreatment did not permit the direct activation of the myofilaments by externally applied Ca2+. The approximately 1000-fold difference in the effective concentrations of ryanodine in intact vs. skinned cardiac cells suggests that low concentrations of ryanodine act in the intact cardiac tissues through processes or on structures that are destroyed by the skinning procedure. No significant differences were observed in the effects of ryanodine in skinned cardiac cells from different adult mammalian species.     

Time and calcium dependence of activation and inactivation of calcium- induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell

Microprocessor-controlled changes of [free Ca2+] at the outer surface of the sarcoplasmic reticulum (SR) wrapped around individual myofibrils of a skinned canine cardiac Purkinje cell and aequorin bioluminescence recording were used to study the mechanism of Ca2+-induced release of Ca2+ from the SR. This Ca2+ release is triggered by a rapid increase of [free Ca2+] at the outer surface of the SR of a previously quiescent skinned cell. Ca2+-induced release of Ca2+ occurred under conditions that prevented any synthesis of ATP from ADP, was affected differentially by interventions that depressed the SR Ca2+ pump about equally, and required ionic conditions incompatible with all known Ca2+-releasing, uncoupled, partial reactions of the Ca2+ pump. Increasing the [free Ca2+]trigger up to an optimum increased the amount of Ca2+ released. A supraoptimum increase of [free Ca2+] trigger inactivated Ca2+-induced release of Ca2+, but partial inactivation was also observed at [free Ca2+] below that necessary for its activation. The amplitude of the Ca2+ release induced by a given increase of [free Ca2+] decreased when the rate of this increase was diminished. These results suggest that Ca2+-induced release of Ca2+ is through a channel across the SR membrane with time- and Ca2+-dependent activation and inactivation. The inactivating binding site would have a higher affinity for Ca2+ but a lower rate constant than the activating site. Inactivation appeared to be a first-order kinetic reaction of Ca2+ binding to a single site at the outer face of the SR with a Q10 of 1.68. The removal of inactivation was the slowest step of the cycle, responsible for a highly temperature-dependent (Q10 approximately 4.00) refractory period.     

Use of aequorin for the appraisal of the hypothesis of the release of calcium from the sarcoplasmic reticulum induced by a change of pH in skinned cardiac cells

A change of pH did not modify the sensitivity of aequorin to Ca2+, but an increase of pH enhanced the Ca2+ sensitivity of the myofilaments of a skinned canine cardiac Purkinje cell. The tension-pCa curve did not present any hysteresis when a given [free Ca2+] was reached from a higher versus from a lower [free Ca2+] in the presence of pH 6.60, 7.10 or 7.40. A rapid variation of pH in either direction failed to induce Ca2+ release from the sarcoplasmic reticulum (SR). The proton ionophores CCCP and gramicidin also failed to induce Ca2+ release from the SR. Increase of pH from 7.10 to 7.40 enhanced Ca2+ accumulation into the SR and, thereby, augmented the Ca2+ content of the SR. Consequently, the amplitude of a subsequent Ca2+ release triggered by a rapid increase of [free Ca2+] at the outer surface of the SR was increased. Conversely, a decrease of pH from 7.10 to 6.60 diminished the Ca2+ accumulation into the SR, the Ca2+ content of the SR and the amplitude of a subsequent Ca2+-induced release of Ca2+ from the SR. In addition, the optimum [free Ca2+] for triggering Ca2+-induced release of Ca2+ was shifted to higher [free Ca2+] values by a decrease of pH from 7.40 to 7.10 or 7.10 to 6.60. This may help to explain the enhancement of the aequorin light transient during acidosis in the intact cardiac muscle inasmuch as acidosis may increase the [free Ca2+] trigger at the outer surface of the SR by inhibiting Na+-Ca2+ exchange across the sarcolemma.     

Excitation-contraction coupling in intact frog skeletal muscle fibers injected with mmolar concentrations of fura-2.

Experiments were carried out to test the hypothesis that mM concentrations of fura-2, a high-affinity Ca2+ buffer, inhibit the release of Ca2+ from the sarcoplasmic reticulum (SR) of skeletal muscle fibers. Intact twitch fibers from frog muscle, stretched to a long sarcomere length and pressure-injected with fura-2, were activated by an action potential. Fura-2's absorbance and fluorescence signals were measured at different distances from the site of fura-2 injection; thus, the myoplasmic free Ca2+ transient (delta [Ca2+]) and the amount and rate of SR Ca2+ release could be estimated at different myoplasmic concentrations of fura-2 ([fura-2T]). At [fura-2T] = 2-3 mM, the amplitude and half-width of delta [Ca2+] were reduced to approximately 25% of the values measured at [fura-2T] less than 0.15 mM, whereas the amount and rate of SR Ca2+ release were enhanced by approximately 50% (n = 5; 16 degrees C). Similar results were observed in experiments carried out at low temperature (n = 2; 8.5-10.5 degrees C). The finding of an enhanced rate of Ca2+ release at 2-3 mM [fura-2T] is opposite to that reported by Jacquemond et al. (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophys. J. 60:867-873) from analogous experiments carried out on cut fibers. In two experiments involving the injection of larger amounts of fura-2, reductions in SR Ca2+ release were observed; however, we were unable to decide whether these reductions were due to [fura-2T] or to some nonspecific effect of the injection itself. These experiments do, however, suggest that if large [fura-2T] inhibits SR Ca2+ release in intact fibers, [fura-2T] must exceed 6 mM to produce an effect comparable to that reported by Jacquemond et al. in cut fibers. Our clear experimental result that 2-3 mM [fura-2T] enhances SR Ca2+ release supports the proposal that delta [Ca2+] triggered by an action potential normally feeds back to inhibit further release of Ca2+ from the SR (Baylor, S.M., and S. Hollingworth. 1988. J. Physiol. [Lond.]. 403:151-192). Our results provide no support for the hypothesis that Ca(2+)-induced Ca2+ release plays a significant role in excitation-contraction coupling in amphibian skeletal muscle.     

Intracellular calibration of the calcium indicator indo-1 in isolated fibers of Xenopus muscle.

Estimates of the free myoplasmic [Ca2+] ([Ca2+]i) with fluorescent dyes are complicated by the fact that some properties of these dyes are altered in the intracellular environment. In the present study indo-1 was used to measure [Ca2+]i in isolated muscle fibers from Xenopus frogs. Fluorescent ratio signals obtained from indo-1 were converted into [Ca2+]i by means of an intracellular calibration method, which involved microinjection of 0.5 M EGTA and 1 M CaCl2 to get the ratio at very low (Rmin) and high (Rmax) [Ca2+], respectively; ratios at intermediate [Ca2+] were obtained by injection of solutions with different EGTA/Ca(2+)-EGTA proportions. This calibration gave an intracellular Ca2+ dissociation constant of indo-1 of 311 nM and a [Ca2+]i at rest of 52 +/- 4 nM (mean +/- SE; n = 15). Indo-1 records during twitches were compared with records obtained with the much faster indicator mag-indo-1. This analysis suggests a Ca2+ dissociation rate of indo-1 of 52 s-1 (22 degrees C). This makes indo-1 less suitable for measurements of [Ca2+]i during twitches, whereas it is fast enough to follow most aspects of [Ca2+]i during tetani, including the relaxation phase.     

Activation of single cardiac and skeletal ryanodine receptor channels by flash photolysis of caged Ca2+.

Single ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels isolated from rabbit skeletal and canine cardiac muscle were reconstituted in planar lipid bilayers. Single channel activity was measured in simple solutions (no ATP or Mg2+) with 250 mM symmetrical Cs+ as charge carrier. A laser flash was used to photolyze caged-Ca2+ (DM-nitrophen) in a small volume directly in front of the bilayer. The free [Ca2+] in this small volume and in the bulk solution was monitored with Ca2+ electrodes. This setup allowed fast, calibrated free [Ca2+] stimuli to be applied repetitively to single SR Ca2+ release channels. A standard photolytically induced free [Ca2+] step (pCa 7-->6) was applied to both the cardiac and skeletal release channels. The rate of channel activation was determined by fitting a single exponential to ensemble currents generated from at least 50 single channel sweeps. The time constants of activation were 1.43 +/- 0.65 ms (mean +/- SD; n = 5) and 1.28 +/- 0.61 ms (n = 5) for cardiac and skeletal channels, respectively. This study presents a method for defining the fast Ca2+ regulation kinetics of single SR Ca2+ release channels and shows that the activation rate of skeletal SR Ca2+ release channels is consistent with a role for CICR in skeletal muscle excitation-contraction coupling.     

Regulation of the ryanodine receptor calcium release channel of the sarcoplasmic reticulum in skeletal muscle

In striated muscle contraction is under the tight control of myoplasmic calcium concentration ([Ca2+]i): the elevation in [Ca2+]i and the consequent binding of calcium to troponin C enables, while the decrease in [Ca2+]i prevents the actin-myosin interaction. Calcium ions at rest are stored in the sarcoplasmic reticulum (SR) from which they are rapidly released upon the depolarisation of the sarcolemmal and transverse (T-) tubular membranes of the muscle cell. The protein responsible for this controlled and fast release of calcium is the calcium release channel found in the membrane of the terminal cisternae of the SR. This review focuses on the physiological and pharmacological modulators of the calcium release channel and tries to draw an up-to-date picture of the events that occur between T-tubular depolarisation and the release of calcium from the SR.     

Measurement of sarcoplasmic reticulum Ca2+ content in intact amphibian skeletal muscle fibres with 4-chloro-m-cresol.

Single skeletal muscle fibres were isolated from the toad (Bufo marinus) and isometric force and myoplasmic free calcium concentration ([Ca2+]i) were measured. Brief applications of 4-chloro- m-cresol (4-CmC, 0.2-5 mM) elevated [Ca2+]i reversibly in a dose-dependent manner. The lowest concentration of 4-CmC which reliably gave maximal [Ca2+]i was 2 mM and it was, therefore, used for measurement of sarcoplasmic reticulum (SR) Ca2+ content. Tetanic stimulations (100 Hz) increased [Ca2+]i from a resting level of 105 +/- 47 nM (n = 10) to 1370 +/- 220 nM (n = 6). Application of 2 mM 4-CmC produced a contracture that was 54 +/- 16% (n = 6) of the tetanic force and elevated [Ca2+]i to a peak of 3520 +/- 540 nM (n = 8). Both force and [Ca2+]i levels (resting and tetanic) were restored after 10 min of washout of 4-CmC. In skinned muscle fibres, the myofibrillar Ca(2+)-sensitivity was not changed by 4-CmC, but maximal force was reduced to 74 +/- 10% (n = 4). The magnitude of the peak of the 4-CmC-induced Ca2+ transient was not significantly changed by removal of extracellular Ca2+ nor by inhibiting the SR Ca2+ pump with 2,5-di-tert-butylhydroquinone. Treatment of intact fibres with 30 mM caffeine produced a peak Ca2+ level that was indistinguishable from 2 mM 4-CmC. These results indicate that it is possible to measure the SR Ca2+ content in the same fibre with 4-CmC without loss of normal muscle function.     

Relationship between myoplasmic calcium transients and calcium currents in frog skeletal muscle

Ca2+ currents (ICa) and myoplasmic Ca2+ transients were simultaneously recorded in single muscle fibers from the semitendinosus muscle of Rana pipiens. The vaseline-gap voltage-clamp technique was used. Ca2+ transients were recorded with the metallochromic indicator dye antipyrylazo III. Ca2+ transients consisted of an early fast rising phase followed by a late slower one. The second phase was increased by experimental maneuvers that enlarged ICa, such as augmenting [Ca2+]o (from 2 to 10 mM) or adding (-)-Bay K 8644 (2 microM). When [Ca2+]o was increased, the second phase of the Ca2+ transients and ICa showed an average increase at 0 mV of 2 +/- 0.9 microM (4) and 1.4 +/- 0.3 mA/ml (4), respectively. (-)-Bay K 8644 increased the late phase of the Ca2+ transients and ICa at 0 mV by 0.8 +/- 0.3 microM (3) and 6.7 +/- 2.0 mA/ml (4), respectively. The initial fast rising phase of the Ca2+ transients was not modified. (-)-Bay K 8644 slowed the time constant of decay of the transients by 57 +/- 6 ms. In other experimental conditions, Ca2+ release from the sarcoplasmic reticulum (SR) was impaired with repetitive stimulation in 1 mM [EGTA]i-containing fibers. Under those circumstances, Ca2+ transients directly followed the time integral of ICa. Pulses to 0 mV caused a large Ca2+ transient that became suppressed when large pulses to 100 mV were applied. In fibers with functioning SR, pulses to 100 mV elicited somewhat smaller or similar amplitude Ca2+ transients when compared with those elicited by pulses to 0 mV. The increase in ICa after raising [Ca2+]o or adding (-)-Bay K 8644 cannot directly explain the change in Ca2+ transients in fibers with functioning SR. On the other hand, when Ca2+ release from the SR is impaired Ca2+ transients depend on ICa.     

Modulation of agonist-induced Ca2+ release by SR Ca2+ load: direct SR and cytosolic Ca2+ measurements in rat uterine myocytes

Release of Ca2+ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca2+ release is considered to be dependent on the Ca2+ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca2+ load on the amplitude of [Ca2+]i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca2+ level ([Ca2+]L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca2+ (10 mM) extracellular solution leads to a substantial increase in [Ca2+]L (SR overload). However, despite increased SR Ca2+ content this did not potentiate ATP-induced [Ca2+]i transients. Repetitive applications of ATP in the absence of extracellular Ca2+, as well as prolonged incubation in Ca2+-free solution without agonist, depleted the [Ca2+]L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca2+ release. ATP-induced [Ca2+]i transients were completely abolished when SR Ca2+ content was decreased below 80% of its normal value indicating a steep dependence of the IP3-mediated Ca2+ release on the Ca2+ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca2+ load below its normal resting level substantially reduces the IP3-mediated Ca2+ release, while Ca2+ overload of the SR has no impact on such release.     

Magnesium permeability of sarcoplasmic reticulum. Mg2+ is not countertransported during ATP-dependent Ca2+ uptake by sarcoplasmic reticulum

Magnesium transport across sarcoplasmic reticulum (SR) vesicles was investigated in reaction mixtures of various composition using antipyrylazo III or arsenazo I to monitor extravesicular free Mg2+. The half-time of passive Mg2+ efflux from Mg2+-loaded SR was 100 s in 100 mM KCl, 150 S in 100 mM K gluconate, and 370 S in either 100 mM Tris methanesulfonate or 200 mM sucrose solutions. The concentration and time course of Mg2+ released into the medium was also measured during ATP-dependent Ca2+ uptake by SR. In reaction mixtures containing up to 3 mM Mg2+, small changes in free magnesium of 10 microM or less were accurately detected without interference from changes in free Ca2+ of up to 100 microM. Three experimental protocols were used to determine whether the increase of free [Mg2+] in the medium after an addition of ATP was due to Mg2+ dissociated from ATP following ATP hydrolysis or to Mg2+ translocation from inside to outside of the vesicles. 1) In the presence of ATP-regenerating systems which maintained constant ATP to ADP ratios and normal rates of active Ca2+ uptake, the increase of Mg2+ in the medium was negligible. 2) Mg2+ released during ATP-dependent Ca2+ uptake by SR was similar to that observed during ATP hydrolysis catalyzed by apyrase, in the absence of SR. 3) In SR lysed with Triton X-100 such that Ca2+ transport was uncoupled from ATPase activity, the rate and amount of Mg2+ release was greater than that observed during ATP-dependent Ca2+ uptake by intact vesicles. Taken together, the results indicate that passive fluxes of Mg2+ across SR membranes are 10 times faster than those of Ca2+ and that Mg2+ is not counter-transported during active Ca2+ accumulation by SR even in reaction mixtures containing minimal concentrations of membrane permeable ions that could be rapidly exchanged or cotransported with Ca2+ (e.g. K+ or Cl-).     

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.     

Reactive disulfides trigger Ca2+ release from sarcoplasmic reticulum via an oxidation reaction

Reactive disulfide compounds (RDSs) with a pyridyl ring adjacent to the S-S bond such as 2,2'-dithiodipyridine (2,2'-DTDP), 4,4'-dithiodipyridine, and N-succinimidyl 3(2-pyridyldithio)propionate (SPDP) trigger Ca2+ release from sarcoplasmic reticulum (SR) vesicles. They are known to specifically oxidize free SH sites via a thiol-disulfide exchange reaction with the stoichiometric production of thiopyridone. Thus, the formation of a mixed S-S bond between an accessible SH site on an SR protein and a RDS causes large increases in SR Ca2+ permeability. Reducing agents, glutathione (GSH) or dithiothreitol reverse the effect of RDSs and permit rapid re-uptake of Ca2+ by the Ca2+, Mg2+-ATPase. The RDSs, 2,2'-DTDP, 4,4'-dithiodipyridine and SPDP displaced [3H]ryanodine binding to the Ca2+-receptor complex at IC50 values of 7.5 +/- 0.2, 1.5 +/- 0.1, and 15.4 +/- 0.1 microM, respectively. RDSs did not alter the rapid initial phase of Ca2+ uptake by the pump, stimulated ATPase activity, and induced release from passively loaded vesicles with nonactivated pumps; thus they act at a Ca2+ release channel and not at the Ca2+, Mg2+-ATPase. Efflux rates increased in 0.25-1.0 mM [Mg2+]free then decreased in 2-5 mM [Mg2+]free. Adenine nucleotides inhibited the oxidation of SHs on SR protein by RDSs and thus reduced Ca2+ efflux rates. However, once RDSs oxidized these SH sites and opened the Ca2+ release pathway, subsequent additions of nucleotides stimulated Ca2+ efflux. In skinned fibers, 2,2'-dithiodipyridine elicited rapid twitches which were blocked by ruthenium red. These results indicate that RDSs trigger Ca2+ release from SR by oxidizing a critical SH group, and thus provide a method to covalently label the protein(s) involved in causing these changes in Ca2+ permeability.     

Perturbation of sarcoplasmic reticulum calcium release and phenol red absorbance transients by large concentrations of fura-2 injected into frog skeletal muscle fibers

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with two indicator dyes: phenol red, to monitor a previously described signal (denoted delta pHapp; Hollingworth and Baylor. 1990. J. Gen. Physiol. 96:473-491) possibly reflective of a myoplasmic pH change following action potential stimulation; and fura-2, to monitor the associated change in the myoplasmic free calcium concentration (delta[Ca2+]). Additionally, it was expected that large myoplasmic concentrations of fura-2 (0.5-1.5 mM) might alter delta pHapp, since it was previously found (Baylor and Hollingworth. 1988. J. Physiol. 403:151-192) that the Ca2(+)-buffering effects of large fura-2 concentrations: (a) increase the estimated total concentration of Ca2+ (denoted by delta[CaT]) released from the sarcoplasmic reticulum (SR), but (b) reduce and abbreviate delta[Ca2+]. The experiments show that delta pHapp was increased at the larger fura-2 concentrations; moreover, the increase in delta pHapp was approximately in proportion to the increase in delta[CaT]. At all fura-2 concentrations, the time course of delta pHapp, through time to peak, was closely similar to, although probably slightly slower than, that of delta[CaT]. These properties of delta pHapp are consistent with an hypothesis proposed by Meissner and Young (1980. J. Biol. Chem. 255:6814-6819) and Somlyo et al. (1981. J. Cell Biol. 90:577-594) that a proton flux from the myoplasm into the SR supplies a portion of the electrical charge balance required as Ca2+ is released from the SR into the myoplasm. A comparison of the amplitude of delta pHapp with that of delta[CaT] indicates that, in response to a single action potential, 10-15% of the charge balance required for Ca2+ release may be carried by protons.     

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.     

Heparin induces Ca2+ release from the terminal cisterns of skeletal muscle sarcoplasmic reticulum

Using a Ca2+-selective electrode and the chlorotetracycline fluorescence technique, the effects of heparin on Ca2+ transport in the sarcoplasmic reticulum (SR) of skeletal muscles in the absence of oxalate were investigated. It was shown that heparin (0.5-10 micrograms/ml) causes a rapid release of 40-50 nmol Ca2+/mg protein from the terminal cistern SR vesicles bound to 130-150 nmol/mg protein of Ca2+ in the presence of ATP. However, heparin has practically no effect on the longitudinal cistern fraction of SR. The effects of heparin can be prevented by ruthenium red. No influence of heparin is observed in the case of the Ca2+-induced release of Ca2+ from the terminal cisterns. When the Ca2+ release is induced by heparin, no Ca2+-induced release of Ca2+ takes place.     

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.     

The luminal Ca2+ transient controls Ca2+ release/re-uptake of sarcoplasmic reticulum

Our recent study (Saiki, Y., and Ikemoto, N., Biochemistry 38, 3112-3119, 1999) suggests that Ca2+ release and re-uptake of the released Ca2+ are coordinated. The following results suggest that the coordination is mediated by the luminal Ca2+ ([Ca2+]lum) transient. Upon inducing the release of the passively loaded Ca2+ from the SR with polylysine, the luminal Ca2+ ([Ca2+]lum) first increased then decreased ([Ca2+]lum transient). The activity of the SR Ca2+ ATPase was monitored at different times after inducing Ca2+ release. The phosphoenzyme (EP) formation as determined by the MANT-fluorescence increased concurrently with the initial rapid increase in the [Ca2+]lum. EP decay (pumping turnover) was accelerated concurrently with a decrease of the [Ca2+]lum. The results suggest that the [Ca2+]lum transient serves as a mediator for the acceleration of the Ca2+ re-uptake occurring soon after the induction of Ca2+ release.