Drug-induced calcium release from heavy sarcoplasmic reticulum of skeletal muscle

Calcium release from isolated heavy sarcoplasmic reticulum of rabbit skeletal muscle by several calmodulin antagonistic drugs was measured spectrophotometrically with arsenazo III and compared with the properties of the caffeine-induced calcium release. Trifluoperazine and W7 (about 500 microM) released all actively accumulated calcium (half-maximum release at 129 microM and 98 microM, respectively) in the presence 0.5 mM MgCl2 and 1 mg/ml sarcoplasmic reticulum protein; calmidazolium (100 microM) and compound 48/80 (70 micrograms/ml) released maximally 30-40% calcium, whilst bepridil (100 microM) and felodipin (50 microM) with calmodulin antagonistic strength similar to trifluoperazine (determined by inhibition of the calcium, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum) did not cause a detectable calcium release, indicating that this drug-induced calcium release is not due to the calmodulin antagonistic properties of the tested drugs. Calcium release of trifluoperazine, W7 and compound 48/80 and that of caffeine was inhibited by similar concentrations of magnesium (half-inhibition 1.4-4.2 mM compared with 0.97 mM for caffeine) and ruthenium red (half-inhibition for trifluoperazine, W7 and compound 48/80 was 0.22 microM, 0.08 microM and 0.63 micrograms/ml, respectively, compared with 0.13 microM for caffeine), suggesting that this drug-induced calcium release occurs via the calcium-gated calcium channel of sarcoplasmic reticulum stimulated by caffeine or channels with similar properties.     

Pathways of calcium release from heavy sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle

The active uptake and efflux of Ca2+ from suspensions of vesicles from heavy rabbit muscle sarcoplasmic reticulum have been examined using the antipyrylazo III dye method in the presence of various nucleotide triphosphate substrates to support active Ca2+ accumulation. On addition of ATP, Ca2+ is rapidly accumulated and maintained at high internal concentrations until the substrate for pump protein is exhausted. Ca2+-induced Ca2+ release which is inhibited by ruthenium red can be demonstrated. The kinetics of Ca2+ release via these channels is different from the Ca2+ efflux observed after substrate exhaustion. This rate was found to be dependent on the type of nucleotide triphosphate, decreasing in the order ATP greater than GTP greater than CTP greater than ITP UTP. It is suggested that different conformations of the Ca2+ pump protein induced by the different substrates may result in the creation of pathways for the facilitated diffusion of Ca2+.     

Quinacrine inhibits the calcium-induced calcium release in heavy sarcoplasmic reticulum vesicles

Quinacrine is a fluorescence probe useful for studying the effect of local anesthetics. The interaction of quinacrine and sarcoplasmic reticulum membranes measured by fluorescence spectroscopy indicates the presence of a saturable binding site. Typical local anesthetics are able to displace quinacrine bound to heavy sarcoplasmic reticulum membranes. The effectiveness of that displacement decreases in the order dibucaine greater than tetracaine greater than benzocaine greater than lidocaine greater than procaine greater than procainamide, indicating that the size and hydrophobicity of quinacrine are major determinants in the binding process. The use of radioactive tracer and a rapid filtration technique reveals that quinacrine interacts, at lower concentrations, with sarcoplasmic reticulum membranes by blocking the Ca2+-induced Ca2+ release. Higher quinacrine concentrations also affect the Ca2+-pump activity.     

Mechanism of anthraquinone-induced calcium release from skeletal muscle sarcoplasmic reticulum

The anthraquinones, doxorubicin, mitoxantrone, daunorubicin and rubidazone are shown to be potent stimulators of Ca2+ release from skeletal muscle sarcoplasmic reticulum (SR) vesicles and to trigger transient contractions in chemically skinned psoas muscle fibers. These effects of anthraquinones are the direct consequence of their specific interaction with the [3H] ryanodine receptor complex, which constitutes the Ca2+ release channel from the triadic junction. In the presence of adenine nucleotides and physiological Mg2+ concentrations (approximately 1.0 mM), channel activation by doxorubicin and daunorubicin exhibits a sharp dependence on submicromolar Ca2+ which is accompanied by a selective, dose-dependent increase in the apparent affinity of the ryanodine binding sites for Ca2+, in a manner similar to that previously reported with caffeine. Unlike caffeine, however, anthraquinones increase [3H]ryanodine receptor occupancy to the level observed in the presence of adenine nucleotides. A strong interaction between the anthraquinone and the caffeine binding sites on the Ca2+ release channel is also observed when monitoring Ca2+ fluxes across the SR. Millimolar caffeine both inhibits anthraquinone-stimulated Ca2+ release, and reduces anthraquinone-stimulated [3H]ryanodine receptor occupancy, without changing the effective binding constant of the anthraquinone for its binding site. The degree of cooperativity for daunorubicin activation of Ca2+ release from SR also increases in the presence of caffeine. These results demonstrate that the mechanism by which anthraquinones stimulate Ca2+ release is caused by a direct interaction with the [3H]ryanodine receptor complex, and by sensitization of the Ca2+ activator site for Ca2+.     

Calcium release from two fractions of sarcoplasmic reticulum from rabbit skeletal muscle

Calcium release from sarcoplasmic reticulum vesicles presumably derived from longitudinal tubules (LSR) and terminal cisternae (HSR) of rabbit skeletal muscle was investigated by dual wavelength spectrophotometry using the calcium-indicator antipyrylazo III. In 120 mM KCl, 5 mM MgCl2, 30 microM, CaCl2, 50 microM MgATP, 100 microM antipyrylazo III, 40 mM histidine (pH 6.8, 25 degrees C), LSR and HSR sequestered approx. 115 nmol calcium/mg, and then spontaneously released calcium. Analysis of ATP hydrolysis and phosphoenzyme level during LSR and HSR calcium sequestration indicated that this calcium release process was passive, occurring in the virtual absence of ATP and phosphoenzyme. Moreover, subsequent addition of ATP reinitiated the calcium sequestration-release sequence. Calcium release by HSR was more than 4-times faster than that by LSR. Analysis of the calcium release phase demonstrated a biexponential decay for both LSR (0.10 and 0.63 min-1) and HSR (0.26 and 1.65 min-1), suggestive of heterogeneity within each fraction. Replacement of 120 mM KCl with either 120 mM choline chloride, 240 mM sucrose, or H2O reduced maximal calcium sequestration by LSR, but had less effect on LSR calcium release rate constants. In the case of HSR, these changes in the ionic composition of the medium drastically reduced calcium release rate constants with little effect on calcium content. These marked differences between LSR and HSR are consistent with the hypothesis that the calcium permeability of the terminal cisternae is greater and more sensitive to the ionic environment than is that of the longitudinal tubules of sarcoplasmic reticulum.     

Quercetin stimulation of calcium release from rabbit skeletal muscle sarcoplasmic reticulum

To elucidate the mechnism by which quercetin enhances the rate of tension development in skinned muscle fibers, effects on calcium release from longitudinal tubule-derived SR (LSR) after phosphate-supported calcium uptake were examined. In all studies, 100 μM quercetin (which inhibits initial calcium uptake velocity 85%) was added at or shortly after the time calcium content reached a maximum at various extravesicular Ca²⁺ concentrations (Ca_o). At moderate Ca_o (0.2–1.0 μM). where spontaneous calcium release rate depended on Ca_o, quercetin caused a marked stimulation of calcium release. This was accompanied by a 60% reduction in calcium influx and a 30-fold increase in calcium efflux. Thus, the previously reported quercetin-induced increase in the rate of tension development by skinned muscle fibers may result, at least in part, from sensitization of Ca²⁺-triggered calcium release to lower Ca_o.     

Mechanism of calcium release from skeletal sarcoplasmic reticulum

Summary Ca²⁺-induced Ca²⁺ release at the terminal cisternae of skeletal sarcoplasmic reticulum was demonstrated using heavy sarcoplasmic reticulum vesicles. Ca²⁺ release was observed at 10 m Ca²⁺ in the presence of 1.25mm free Mg²⁺ and was sensitive to low concentrations of ruthenium red and was partially inhibited by valinomycin. These results suggest that the Ca²⁺-induced Ca²⁺ release is electrogenic and that an inside negative membrane potential created by the Ca²⁺ flux opens a second channel that releases Ca²⁺. Results in support of this formulation were obtained by applying a Cl^– gradient or K⁺ gradient to sarcoplasmic reticulum vesicles to initiate Ca²⁺ release. Based on experiments the following hypothesis for the excitation-contraction coupling of skeletal muscle was formulated. On excitation, small amounts of Ca²⁺ enter from the transverse tubule and interact with a Ca²⁺ receptor at the terminal cisternae and cause Ca²⁺ release (Ca²⁺-induced Ca²⁺ release). This Ca²⁺ flux generates an inside negative membrane potential which opens voltage-gated Ca²⁺ channels (membrane potential-dependent Ca²⁺ release) in amounts sufficient for contraction.     

G-protein dependent potentiation of calcium release from sarcoplasmic reticulum of skeletal muscle

Skinned fibre experiments were conducted to determine if guanine nucleotide-binding proteins play a role in excitation-contraction coupling of skeletal muscle. By itself, the GTP-gamma S, a non hydrolysable GTP analogue was unable to induce calcium release from the sarcoplasmic reticulum, even at concentrations as high as 500 microM. However, calcium- or caffeine-induced calcium releases were enhanced by GTP-gamma S in micromolar concentrations. This response was blocked by GDP-beta S or Pertussis toxin. 32P-ADP-ribosylation catalysed by Pertussis toxin, radiolabelled G-protein alpha subunits in the range of 40 kDa on membrane subcellular fractions of rat skeletal muscle. Using Western blot analysis with antibodies raised against the bovine transducin, G-proteins were identified in frog and rat skeletal muscle subcellular fractions. In most of the muscle fractions (plasma membrane, T-tubules, triads, sarcoplasmic reticulum), the anti-beta subunit antibodies recognized a 36 kDa protein which comigrated with transducin beta subunit. It appears therefore that some of the G-proteins identified by ADP-ribosylation or immunostaining in several subcellular fractions from skeletal muscle, are implicated in the modulation of calcium release from sarcoplasmic reticulum. These results suggest that a Pertussis toxin sensitive G-protein is present at the loci of E-C coupling, and that it serves to regulate the calcium release.     

Phosphatidylinositol 4,5-bisphosphate enhances calcium release from sarcoplasmic reticulum of skeletal muscle

In the course of our study on the function of sarcoplasmic reticulum (SR) in skeletal muscle, the stimulatory action of phosphatidylinositol 4,5-bisphosphate (PIP2) on the Ca2+ release from SR was demonstrated by using chemically skinned fibers and fragmented SR vesicles. PIP2 induced a tension spike followed by sustained contraction in skinned fibers. PIP2 enhanced the caffeine-induced Ca2+ release from SR vesicles at low concentrations and triggered Ca2+ release by itself at high concentrations. PIP2 also enhanced 45Ca2+ efflux from SR vesicles. However, inositol 1,4,5-triphosphate never produced these effects. The Ca2+-releasing action of PIP2 was only weakly affected by ruthenium red or procaine. These observations suggest that PIP2 activates an SR Ca2+ release channel whose properties are different from those of the Ca2+-induced Ca2+ release channel.     

Uptake and release of calcium ions by heavy sarcoplasmic reticulum fraction of normal and malignant hyperthermia-susceptible human skeletal muscle

• 1.1. Ca²⁺ uptake, Ca²⁺-dependent ATPase activity and halothane-induced Ca²⁺ release from the heavy sarcoplasmic reticulum fraction of muscle from malignant hyperthermia susceptible individuals are similar to those of normal human muscle.
• 2.2. Ca²⁺-induced Ca²⁺ release from the diseased muscle was increased by 13%.

     

Ethanol increases calcium permeability of heavy sarcoplasmic reticulum of skeletal muscle

The effects of ethanol on both Ca2+ pump activity and Ca2+-induced Ca2+ release in sarcoplasmic reticulum (SR) of rabbit skeletal muscle were studied. In concentrations of 0.1-1.0%, ethanol conspicuously enhanced Ca2+ release from the heavy fraction of SR, whereas a much smaller effect was noted in the light fraction. When Ca2+-induced Ca2+ release was inhibited by 10 mM Mg2+, the Ca2+ pump activities of both the heavy and light SR were the same; the activities were not significantly influenced by 1% ethanol. Ethanol itself did not release Ca2+ from the heavy SR. However, it appeared to render the heavy SR more permeable to Ca2+, thereby decreasing the amount of storable Ca2+. This action of ethanol may be related to the mechanism of its negative inotropic effect.     

Enhanced Ca2+-induced calcium release by isolated sarcoplasmic reticulum vesicles from malignant hyperthermia susceptible pig muscle

To further define the possible involvement of sarcoplasmic reticulum calcium accumulation and release in the skeletal muscle disorder malignant hyperthermia (MH), we have examined various properties of sarcoplasmic reticulum fractions isolated from normal and MH-susceptible pig muscle. A sarcoplasmic reticulum preparation enriched in vesicles derived from the terminal cisternae, was further fractionated on discontinuous sucrose density gradients (Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374). The resultant MH-susceptible and normal sarcoplasmic reticulum fractions, designated F0-F4, did not differ in yield, cholesterol and phospholipid content, or nitrendipine binding capacity. Calcium accumulation (0.27 mumol Ca/mg per min at 22 degrees C), Ca2+-ATPase activity (0.98 mumol Pi/mg per min at 22 degrees C), and calsequestrin content were also similar for MH-susceptible and normal sarcoplasmic reticulum fraction F3. To examine sarcoplasmic reticulum calcium release, fraction F3 vesicles were passively loaded with 45Ca (approx. 40 nmol Ca/mg), and rapidly diluted into a medium of defined Ca2+ concentration. Upon dilution into 1 microM Ca2+, the extent of Ca2+-dependent calcium release measured after 5 s was significantly greater for MH-susceptible than for normal sarcoplasmic reticulum, 65.9 +/- 2.8% vs. 47.7 +/- 3.9% of the loaded calcium, respectively. The C1/2 for Ca2+ stimulation of this calcium release (5 s value) from MH-susceptible sarcoplasmic reticulum also appeared to be shifted towards a higher Ca2+-sensitivity when compared to normal sarcoplasmic reticulum. Dantrolene had no effect on calcium release from fraction F3, however, halothane (0.1-0.5 mM) increased the extent of calcium release (5 s) similarly in both MH-susceptible and normal sarcoplasmic reticulum. Furthermore, Mg2+ was less effective at inhibiting, while ATP and caffeine were more effective in stimulating, this Ca2+-dependent release of calcium from MH-susceptible, when compared to normal sarcoplasmic reticulum. Our results demonstrate that while sarcoplasmic reticulum calcium-accumulation appears unaffected in MH, aspect(s) of the sarcoplasmic reticulum Ca2+-induced calcium release mechanism are altered. Although the role of the Ca2+-induced calcium release mechanism of sarcoplasmic reticulum in situ is not yet clear, our results suggest that an abnormality in the regulation of sarcoplasmic reticulum calcium release may play an important role in the MH syndrome.     

Hypochlorous acid modifies calcium release channel function from skeletal muscle sarcoplasmic reticulum.

We have previously demonstrated that H2O2 at millimolar concentrations induces Ca(2+) release from actively loaded sarcoplasmic reticulum (SR) vesicles and induces biphasic [(3)H]ryanodine binding behavior. Considering that hypochlorous acid (HOCl) is a related free radical and has been demonstrated to be a more effective oxidant of proteins, we evaluated the effects of HOCl on sarcoplasmic reticulum Ca(2+)-channel release mechanism. In a concentration-dependent manner, HOCl activates the SR Ca(2+) release channel and induces rapid release of Ca from actively loaded vesicles. HOCl-induced Ca(2+) release is inhibited in the presence of millimolar concentrations of DMSO. High-affinity [(3)H]ryanodine binding is also enhanced at concentrations from 10 to 100 microM. At HOCl concentrations of >100 microM, equilibrium binding is inhibited. HOCl stimulation of binding is inhibited by the addition of dithiothreitol. The direct interaction between HOCl and the Ca(2+) release mechanism was further demonstrated in single-channel reconstitution experiments. HOCl, at 20 microM, activated the Ca(2+) release channel after fusion of a SR vesicle to a bilayer lipid membrane. At 40 microM, Ca(2+)-channel activity was inhibited. Pretreatment of SR vesicles with HOCl inhibited the fluorescence development of a fluorogenic probe specific to thiol groups critical to channel function. These results suggest that HOCl at micromolar concentrations can modify SR Ca(2+) handling.     

Photooxidation of skeletal muscle sarcoplasmic reticulum induces rapid calcium release.

The photooxidizing xanthene dye rose bengal is shown to induce rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum (SR) vesicles. In the presence of light, nanomolar concentrations of rose bengal increase the Ca2+ permeability of the SR and stimulate the production of singlet oxygen (1O2). In the absence of light, no 1O2 production is measured. Under these conditions, higher concentrations of rose bengal (micromolar) are required to stimulate Ca2+ release. Furthermore, removal of oxygen from the release medium results in marked inhibition of the light-dependent reaction rate. Rose bengal-induced Ca2+ release is relatively insensitive to Mg2+. At nanomolar concentrations, rose bengal inhibits [3H]ryanodine binding to its receptor. beta,gamma-Methyleneadenosine 5'-triphosphate, a nonhydrolyzable analog of ATP, inhibits rose bengal-induced Ca2+ release and prevents rose bengal inhibition of [3H]ryanodine binding. Ethoxyformic anhydride, a histidine modifying reagent, at millimolar concentrations induces Ca2+ release from SR vesicles in a manner similar to that of rose bengal. The molecular mechanism underlying rose bengal modification of the Ca2+ release system of the SR appears to involve a modification of a histidyl residue associated with the Ca2+ release protein from SR. The light-dependent reaction appears to be mediated by singlet oxygen.     

Dantrolene does not block calcium pulse-induced calcium release from a putative calcium channel in sarcoplasmic reticulum from malignant hyperthermia and normal pig muscle

Calcium pulse additions to isolated SR membranes can cause a reversible efflux of calcium. The threshold level of calcium loading at which calcium efflux occurs is lower for SR membranes isolated from malignant hyperthermia susceptible (MHS) swine. Dantrolene, a unique muscle relaxant, had no effect on threshold calcium load, amounts and rates of calcium release from SR isolated from control and MHS skeletal muscle. It is concluded that the putative calcium channel through which this calcium pulse-induced calcium release mechanism occurs is not affected by dantrolene under these experimental conditions.     

Time course of activation of calcium release from sarcoplasmic reticulum in skeletal muscle.

Myoplasmic free calcium transients were measured with antipyrylazo III in voltage clamped segments of frog skeletal muscle fibers and were used to calculate the rate of release (Rrel) of calcium from the sarcoplasmic reticulum. Intramembrane charge movement was measured for the same pulses in the same fibers. During a depolarizing pulse Rrel rose to an early peak and then decayed relatively rapidly but incompletely due to calcium-dependent inactivation (Schneider M.F., and B.J. Simon. 1988. J. Physiol. (Lond.). 405:727-745). Two approaches were used to determine release activation independent of the effects of inactivation: (a) a mathematical correction based on the assumption that inactivation was a process occurring in parallel with and independently of activation; (b) an experimental procedure in which release was maximally inactivated by a large short prepulse and then the remaining noninactivatable component of release was monitored during a subsequent test pulse. Both procedures gave the same time course of activation of release. Release activation paralleled the time course of intramembrane charge movement but was delayed by a few milliseconds.     

Characterization of the tetraphenylboron-induced calcium release from skeletal sarcoplasmic reticulum

Release of Ca2+ from skeletal sarcoplasmic reticulum vesicles was studied by the spectrophotometric stopped-flow technique using tetraphenylboron as a releasing agent. The extent of Ca2+ release shows a sigmoidal response, with respect to the tetraphenylboron concentration, being dependent on Ca2+ preloading and Ca2+-ATPase activity, since these experiments were performed on actively loaded vesicles. The release process has a rapid component with an apparent rate constant of 6-8 s-1, showing a linear relationship between the rapid rate of Ca2+ release and the Ca2+ content of the vesicles. The release is not mediated by the reversal of the Ca2+ pump. Since the amphipathic anion tetraphenylboron was unable to elicit a Ca2+-release response when added to a preparation of sarcoplasmic reticulum phospholipid vesicles, it is suggested that there may be an interaction with some membrane protein(s) at the hydrophobic/hydrophilic interface leading to the opening of some specific Ca2+-release pathway.     

Kinetic studies of calcium-induced calcium release in cardiac sarcoplasmic reticulum vesicles

Fast Ca(2+) release kinetics were measured in cardiac sarcoplasmic reticulum vesicles actively loaded with Ca(2+). Release was induced in solutions containing 1.2 mM free ATP and variable free [Ca(2+)] and [Mg(2+)]. Release rate constants (k) were 10-fold higher at pCa 6 than at pCa 5 whereas Ryanodine binding was highest at pCa < or =5. These results suggest that channels respond differently when exposed to sudden [Ca(2+)] changes than when exposed to Ca(2+) for longer periods. Vesicles with severalfold different luminal calcium contents exhibited double exponential release kinetics at pCa 6, suggesting that channels undergo time-dependent activity changes. Addition of Mg(2+) produced a marked inhibition of release kinetics at pCa 6 (K(0.5) = 63 microM) but not at pCa 5. Coexistence of calcium activation and inhibition sites with equally fast binding kinetics is proposed to explain this behavior. Thimerosal activated release kinetics at pCa 5 at all [Mg(2+)] tested and increased at pCa 6 the K(0.5) for Mg(2+) inhibition, from 63 microM to 136 microM. We discuss the possible relevance of these results, which suggest release through RyR2 channels is subject to fast regulation by Ca(2+) and Mg(2+) followed by time-dependent regulation, to the physiological mechanisms of cardiac channel opening and closing.     

Calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum in skeletal muscle fibers

The steady-state calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum was studied in voltage-clamped, cut segments of frog skeletal muscle fibers containing two calcium indicators, fura-2 and anti-pyrylazo III (AP III). Fura-2 fluorescence was used to monitor resting calcium and relatively small calcium transients during small depolarizations. AP III absorbance signals were used to monitor larger calcium transients during larger depolarizations. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum was calculated from the calcium transients. The equilibrium calcium dependence of inactivation of calcium release was determined using 200-ms prepulses of various amplitudes to elevate [Ca2+] to various steady levels. Each prepulse was followed by a constant test pulse. The suppression of peak Rrel during the test pulse provided a measure of the extent of inactivation of release at the end of the prepulse. The [Ca2+] dependence of inactivation indicated that binding of more than one calcium ion was required to inactivate each release channel. Half-maximal inactivation was produced at a [Ca2+] of approximately 0.3 microM. Variation of the prepulse duration and amplitude showed that the suppression of peak release was consistent with calcium-dependent inactivation of calcium release but not with calcium depletion. The same calcium dependence of inactivation was obtained using different amplitude test pulses to determine the degree of inactivation. Prepulses that produced near maximal inactivation of release during the following test pulse produced no suppression of intramembrane charge movement during the test pulse, indicating that inactivation occurred at a step beyond the voltage sensor for calcium release. Three alternative set of properties that were assumed for the rapidly equilibrating calcium-binding sites intrinsic to the fibers gave somewhat different Rrel records, but gave very similar calcium dependence of inactivation. Thus, equilibrium inactivation of calcium release appears to be produced by rather modest increases in [Ca2+] above the resting level and in a steeply calcium-dependent manner. However, the inactivation develops relatively slowly even during marked elevation of [Ca2+], indicating that a calcium-independent transition appears to occur after the initial calcium-binding step.     

Fatty acid effects on calcium influx and efflux in sarcoplasmic reticulum vesicles from rabbit skeletal muscle

Low concentrations of fatty acids inhibited initial Ca uptake by sarcoplasmic reticulum vesicles, the extent of inhibition varying with chain length and unsaturation in a series of C₁₄–C₂₀ fatty acids. Oleic acid was a more potent inhibitor of initial Ca uptake than stearic acid at 25°C, whereas at 5°C there was less difference between the inhibitory effects of low concentrations of these fatty acids. When the fatty acids were added later, during the phase of spontaneous Ca release that follows Ca uptake in reactions carried out at 25°C, 1–4 μM oleic and stearic acids caused Ca content to increase. This effect was due to marked inhibition of Ca efflux and slight stimulation of Ca influx. At concentrations of >4 μM, both fatty acids inhibited the Ca influx that occurs during spontaneous Ca release; in the case of oleic acid, this inhibition resembled that of initial Ca uptake at 5°C. The different effects of fatty acids at various times during Ca uptake reactions may be explained in part if alterations in the physical state of the membranes occur during the transition from the phase of initial Ca uptake to that of spontaneous Ca release.