Two kinds of calcium-induced release of calcium from the sarcoplasmic reticulum of skinned cardiac cells.

This article compares the Ca(2+)-induced release of Ca2+ that is triggered by a rapid increase of free Ca2+ concentration at the outer surface of the sarcoplasmic reticulum of a skinned cardiac cell to the spontaneous release of Ca2+ that is produced by a steady-state high free Ca2+ concentration which overloads the sarcoplasmic reticulum with Ca2+ in a skinned cardiac cell. The first process, that is triggered by a rapid increase of free Ca2+ concentration at the outer surface of the sarcoplasmic reticulum, has a time- and Ca(2+)-dependent activation and inactivation, does not require any preload of the sarcoplasmic reticulum with Ca2+, and is not affected by the addition of inositol (1,4,5)-trisphosphate. The second process, i.e. the spontaneous release of Ca2+ from the sarcoplasmic reticulum, is not inactivated by a high free Ca2+ concentration, requires an overload of the sarcoplasmic reticulum with Ca2+ and is enhanced by inositol(1,4,5)-trisphosphate. The filling inside the sarcoplasmic reticulum with Ca2+ is critical for the triggering of the spontaneous release of Ca2+. On the other hand, the spontaneous release of Ca2+ has many similarities to the "Ca(2+)-induced release of Ca2+" that is observed for isolated sarcoplasmic reticulum vesicles incorporated into a lipid bilayer which is triggered by the increase of free Ca2+ concentration at the outer surface of the sarcoplasmic reticulum. Although the Ca(2+)-induced release of Ca2+ with time- and Ca(2+)-dependent activation and inactivation and the spontaneous release of Ca2+ are regulated by difference mechanisms, they are both inhibited by ryanodine, which suggests that they may take place through the same channel and may even have some partial common pathway.

Model of calcium-induced calcium release mechanism in cardiac cells.

A model with which to elucidate the mechanism of Ca(2+) release from, and Ca(2+) loading in the sarcoplasmic reticulum (SR) by Ca(2+) current (I Ca) in cardiac cells is proposed. The SR is assumed to be comprised of three functional subcompartments: (1) the main calcium store (MCS), which contains most of the calcium (both free and bound); (2) the releasable terminal (RT), which contains the calcium readily available for release; and (3) the longitudinal network of the SR (LSR), which sequesters and the transfers the sarcoplasmic calcium to the RT. A rapid increase of the Ca(2+) concentration at the outer surface of the SR (Cae) due to the fast component of I(Ca) activates and inactivates this surface, inducing the release of Ca(2+) from the RT to the sarcoplasmic space. The RT in turn is further activated and inactivated by a increase in the concentration of sarcoplasmic Ca(2+). The Ca(2+) in the sarcoplasmic space is then sequestered by the LSR, leading to the reactivation of the RT. Further increase of Cae due to the slow component of I(Ca) enhances the entry of Ca(2+) into the MCS to be bound by the binding substance. The free Ca(2+) released from the Ca-binding substance complex is transferred to the RT for subsequent release. The activation, inactivation and reactivation are Ca(2+)-mediated and time-dependent. The proposed model yields simulation of the many events qualitatively similar to those observed experimentally in skinned cardiac cells.

Appraisal of the physiological relevance of two hypothesis for the mechanism of calcium release from the mammalian cardiac sarcoplasmic reticulum: calcium-induced release versus charge-coupled release.

Recent studies correlating the calcium current with, respectively, the clamp-imposed voltage and the calcium current in intact isolated mammalian cardiac myocytes are reviewed. The major findings are the following: With the exception of one group, all investigators agree that a calcium transient is never observed in the absence of a calcium current. In addition, there is a good correlation between voltage dependence of the calcium current and that of the calcium transient, although this correlation may vary among the cardiac tissues from different animal species. Repolarization clamp pulses from highly positive potentials produce a 'tail current' which is associated with a 'tail calcium transient'. The calcium transient is inhibited when the calcium current is blocked by calcium deprivation or substitution, or by the addition of calcium current antagonists, despite the fact that sarcoplasmic reticulum still contains calcium that can be released by caffeine (with inhibition of this release by ryanodine). These three findings are strongly in favor of a calcium-induced release of calcium and against the hypothesis of charge-movement-coupled release of calcium from the sarcoplasmic reticulum. The only finding that would be more in favor of the latter hypothesis (although still reconciliable with the former) is that repolarization occurring before the rapid rise of calcium transient is complete curtails the calcium transient. Thus, the possibility that charge movement might somehow regulate calcium-induced release of calcium cannot be excluded.

Binding of inositol trisphosphate by a liver microsomal fraction.

Accumulating evidence suggests that the increase in cytosolic Ca2+ induced by receptor agonists is mediated by inositol 1,4,5-trisphosphate, a product of phospholipase C-mediated breakdown of phosphatidylinositol 4,5-bisphosphate. The present study employs inositol tris[32P]phosphate to demonstrate a specific receptor binding site in a microsomal fraction of rat liver.

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.

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.

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.

Simulated calcium current can both cause calcium loading in and trigger calcium release from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell.

Skinned canine cardiac Purkinje cells were stimulated by regularly repeated microinjection-aspiration sequences that were programmed to simulate the fast initial component of the transsarcolemmal Ca2+ current and the subsequent slow component corresponding to noninactivating Ca2+ channels. The simulated fast component triggered a tension transient through Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum (SR). The simulated slow component did not affect the tension transient during which it was first introduced but it potentiated the subsequent transients. The potentiation was not observed when the SR function had been destroyed by detergent. The potentiation decreased progressively when the slow component was separated by an increasing time interval from the fast component. The potentiation was progressive over several beats under conditions that decreased the rate of Ca2+ accumulation into the SR (deletion of calmodulin from the solutions; a decrease of the temperature from 22 to 12 degrees C). In the presence of a slow component, an increase of frequency caused a positive staircase, and the introduction of an extrasystole caused a postextrasystolic potentiation. There was a negative staircase and no postextrasystolic potentiation in the absence of a slow component. These results can be explained by a time- and Ca2+-dependent functional separation of the release and accumulation processes of the SR, rather than by Ca2+ circulation between anatomically distinct loading and release compartments. The fast initial component of transsarcolemmal Ca2+ current would trigger Ca2+ release, whereas the slow component would load the SR with an amount of Ca2+ available for release during the subsequent tension transients.

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.

Spontaneous versus triggered contractions of "calcium-tolerant" cardiac cells from the adult rat ventricle.

Cardiac cells were isolated from the adult rat ventricle by an enzymatic treatment. The cells considered intact were quiescent in the presence of 2.5 mM free Ca2+ but responded to an electrical stimulation by an homogeneous and brief contraction. When the procedure failed, spontaneous cyclic contractions occurred. Often they propagated as a wave from an intercalated disk, and the tension recording showed several components in each contraction. Electrical stimulation at a frequency higher than that of the spontaneous contractions induced synchronous activation with a single component of the tension. Experiments in skinned cardiac cells suggested that the spontaneous cyclic contractions observed in enzymatically separated cardiac cells are caused by a spontaneous cyclic release of Ca2+ from the sarcoplasmic reticulum (SR). This spontaneous release requires a Ca2+ overload of the SR. Its mechanism is different from that of the Ca2+-induced release of Ca2+, which is elicited by a rapid increase of [free Ca2+] at the outer surface of the SR of a previously quiescent skinned cell.

Calcium pools in saponin-permeabilized guinea pig hepatocytes.

The plasma membranes of isolated guinea pig hepatocytes were made permeable with saponin. The cells were then suspended in a medium resembling cytosol in which the level of ATP was kept constant with an ATP-regenerating system. Intracellular ATP-dependent 45Ca and 40Ca sequestration was then followed at various concentrations of Ca2+ in the medium. It was found that ATP-dependent Ca uptake could be divided into two mechanisms: a low affinity high capacity uptake sensitive to 2,4-dinitrophenol (DNP) and oligomycin, thought to be mitochondrial, and a low capacity high affinity uptake, which was insensitive to DNP and oligomycin, thought to be mainly endoplasmic reticulum (ER). The threshold for ATP-dependent Ca uptake by the latter pool was about 20 nM Ca2+. The process had an EC50 value of 0.3 microM (for 45Ca) and a capacity of 2.7 nmol/45Ca/mg of protein. The "ER" mechanism also had a high affinity for ATP (EC50, about 43 microM). There was no significant accumulation of Ca by the postulated mitochondrial pool until the [Ca2+] of the medium was greater than 1 microM. The concentration of Ca2+ in the cytosol of normal unstimulated hepatocytes was estimated from measurements of phosphorylase a activity to be about 0.18 microM. At this [Ca2+], the ER pool of the saponin-treated hepatocytes accumulated Ca but there was no evidence of any Ca uptake into the "mitochondrial" pool. This suggests that most of the exchangeable Ca in a normal cell may be in DNP and oligomycin-insensitive pools (presumably the ER or possibly the plasma membrane) and suggests that these pools are likely to be involved in the increase in cytosolic [Ca2+] which occurs after stimulation by Ca-mobilizing hormones.

Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum.

The hypothesis of a Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is supported by experiments done in skinned cardiac cells (sarcolemma removed by microdissection). According to this hypothesis, the transsarcolemmal Ca2+ influx does not activate the myofilaments directly but through the induction of a Ca2+ release from the SR. The stimulus gating CICR is not a small change in free Ca2+ concentration (delta[free Ca2+]) outside the SR but a function of the rate of this change (delta[free Ca2+/delta t]). The initial relatively fast component of the transsarcolemmal Ca2+ current would trigger Ca2+ release; the subsequent slow component, perhaps corresponding to noninactivating Ca2+ channels, would load the SR with an amount of Ca2+ available for release during subsequent beats. Inactivation of CICR is caused by the large increase of [free Ca2+] outside the SR resulting from Ca2+ release, which inhibits further release. This negative feedback helps to explain that CICR is not all or none. During relaxation the Ca2+ reaccumulation in the SR is backed up by the Ca2+ efflux across the sarcolemma through Na+-Ca2+ exchange and the sarcolemmal Ca2+ pump. Computations of the Ca2+ buffering in the mammalian ventricular cell and of the systolic transsarcolemmal Ca2+ influx do not support the alternative hypothesis that this influx of Ca2+ is large enough to activate the myofilaments directly. Yet the hypothesis of a CICR can be challenged because of many problems and uncertainties related to the preparations and methods used for skinned cardiac cell experiments.

Calcium release in skinned cardiac cells: variations with species, tissues, and development.

The Ca2+-induced release of Ca2+ from he sarcoplasmic reticulum (SR) is not an all-or-none process but is graded with 1) the level of preload of the SR with Ca2+, 2) the rate of change of [free CA2+], and 3) the level of [free CA2+] used as a trigger. Among adult ventricular cardiac tissues the Ca2+-induced release of Ca2+ is absent in the frog ventricle, but present in avian and all mammalian ventricular tissues studied with large variations among species and maximum prominence in the rat ventricle. During the development of the rat ventricular cell there is a progressive maturation of the Ca2+-induced release of Ca2+, which is absent before birth. In the same animal species the Ca2+-induced release of Ca2+ is more developed in the atrium than in the ventricle. In the dog cardiac Purkinje tissue a higher [free Ca2+] preload and a higher [free Ca2+] trigger are required than for the dog ventricle. A working hypothesis is suggested according to which the mechanism of excitation-contraction coupling increases in complexity as the diameter of the cell increases, with maximum complexity presented by the fast skeletal muscle cell.

Fluorescence and differential light absorption recordings with calcium probes and potential-sensitive dyes in skinned cardiac cells.

This report describes an optical system for microspectrophotometry in a single cardiac cell from which the sarcolemma has been removed by microdissection (skinned cardiac cell). This system is attached to the high power inverted microscope used for the microdissection and includes (a) a single variable wavelength microspectrophotometer used to define the spectrum of a given dye or Ca2+ probe; and (b) a dual wavelength, differential microspectrophotometer used to record differentially between the optimum wavelength and a wavelength separated by 25--30 nm. Results are presented using the following optical methods: (a) fluorescence measurements with chlorotetracycline to monitor the amount of Ca2+ bound to the inner face of the sarcoplasmic reticulum (SR) membrane; (b) differential absorption measurements with arsenazo III to measure changes of myoplasmic [Ca2+]free resulting from Ca2+ release from the SR; (c)fluorescence and (or) differential absorption measurements with the potential-sensitive dyes merocyanine 540, NK 2367, and di-S-C3(5) to monitor changes of charge distribution on the SR membrane during Ca2+ accumulation in the SR, as well as before and during Ca2+-induced release of Ca2+ from the SR. A small and rapid signal is observed which precedes the Ca2+-induced release of Ca2+ from the SR. It is detected as an increase of CA2+ binding inside the SR with chlorotetracycline and as a "hyperpolarization" with potential-sensitive dyes, while no transient change of myoplasmic [Ca2+]free is detected with arsenazo III. This small and rapid signal preceding the Ca2+ release may be a first hint to an understanding of the mechanism whereby a small increase of [Ca2+]free outside the SR triggers Ca2+ release from the SR.

A dual-channel signal averager for spontaneous signals.

This signal averager has been developed for spontaneous transients of tension and of aequorin bioluminescence in skinned cardiac cells. The signal with the largest signal-to-noise ratio (generally the tension transient) is entered in Channel 1 and triggers the system. Then the averager will unmask the signal entered in Channel 2 if it was buried in the noise during the direct recording. This signal averager uses an inexpensive microcomputer and a program written in Assembly Language. To facilitate its use, the averager is remotely controlled by a potentiometer for the adjustment of the threshold voltage above which the signal in Channel 1 will be collected and by three push buttons for starting the data collection, for averaging and displaying the results, and for resetting the system. The display is purely analog on the recorder, including averaged signals and interval for the signals in both channels together with a graphical representation of the standard deviations and of the number of observations. This signal averager could be used for any preparations developing spontaneous signals with variable intervals, provided that at least one of the synchronous signals has a large enough signal-to-noise ratio to trigger the system.