Calcium-induced calcium release mechanism from the sarcoplasmic reticulum in skinned crab muscle fibres

Mechanically skinned skeletal muscle fibres of the crab Carcinus maenas have been used to investigate the mechanism of calcium release from the sarcoplasmic reticulum. Calcium release has been monitored by the amplitude and kinetics of the tension developed by the fibre. Results show that a very low calcium concentration, insufficient to directly activate contractile proteins, induces a release of calcium from the SR. This release is stimulated by low concentrations of caffeine and inhibited by small amounts of EGTA. Thus, a graded calcium-induced calcium release mechanism dependent on extrareticular calcium concentration has been demonstrated in skinned crab muscle fibre.     

Characteristics of functioning of electromechanical coupling in striated muscles of higher and lower vertebrates

A comparative pharmacological analysis of relative contributions of different signal transduction pathways in the activation of contraction (excitation-contraction coupling, ECC) in intact fast striated muscles of frog and lamprey was performed. It was found that the major mechanism responsible for the ECC in muscles of both animals is Ca2+ release from the sarcoplasmic reticulum through the ryanodine-sensitive channels. However, the ECC in lamprey muscle displays some important differences in the units of electromechanical coupling, which precede the calcium release from sarcoplasmic reticulum. The maximum contraction force in frog muscle develops during caffeine-induced contracture, which indicates that all Ca2+ stored in sarcoplasmic reticulum is released through ryanodine-sensitive channels. In contrast, in lamprey muscle, the maximum force develops not in response to high caffeine concentration, but in response to repetitive electrical stimulation. Hence, in addition to stores liberated by ryanodine-sensitive channels, some other sources of calcium ions should exist, which contribute to the contraction activation. A source of this additional Ca2+ ions can be external medium, because acetylcholine contracture is abolished in a calcium-free medium. In frog muscle, the acetylcholine contracture was abolished in a Na(+)-free solution. It was concluded that in frog muscle ECC can be triggered by changes in the transmembrane potential (depolarization-induced calcium release), while in lamprey muscle the entry of calcium ions into myoplasm as the trigger in ECC (calcium-induced calcium release). The lamprey muscle was found to be more resistant to tetrodotoxin and tetracaine, which is indicative of a role in the activation of contraction of tetrodotoxin-resistant Na+ and/or Ca2+ channels. It was concluded, that ECC mechanism in striated muscles of low vertebrates is not limited by the generally accepted scheme of depolarization-induced calcium release but can include some other schemes, which require the Ca2+ influx into the cell.     

Role of the different calcium sources in the excitation-contraction coupling in crab muscle fibers

Excitation-contraction coupling in crab muscle fibers was studied in voltage-clamp conditions. Extracellular calcium is essential for the mechanical activity. Two calcium influxes induced by membrane depolarization contribute to tension development: one is the inward calcium current responsible for the phasic tension, the other is a calcium influx dependent on extracellular sodium and calcium concentrations and is responsible for the tonic tension. These calcium influxes are not sufficient to activate contractile proteins. Experiments with procaine and caffeine show that a calcium release from the sarcoplasmic reticulum is required.     

The effect of trifluoroperazine on the sarcoplasmic reticulum membrane

The inhibitory effect of trifluoroperazine (25-200 microM) on the sarcoplasmic reticulum calcium pump was studied in sarcoplasmic reticulum vesicles isolated from skeletal muscle. It was found that the lowest effective concentrations of trifluoroperazine (10 microM) displaces the Ca2+ dependence of sarcoplasmic reticulum ATPase to higher Ca2+ concentrations. Higher trifluoroperazine concentrations (100 microM) inhibit the enzyme even at saturating Ca2+. If trifluoroperazine is added to vesicles filled with calcium in the presence of ATP, inhibition of the catalytic cycle is accompanied by rapid release of accumulated calcium. ATPase inhibition and calcium release are produced by identical concentrations of trifluoroperazine and, most likely, by the same enzyme perturbation. These effects are related to partition of trifluoroperazine ino the sarcoplasmic reticulum membrane, and consequent alteration of the enzyme assembly within the membrane structure, and of the bilayer surface properties. The effect of trifluoroperazine was also studied on dissociated ('chemically skinned') cardiac cells undergoing phasic contractile activity which is totally dependent on calcium uptake and release by sarcoplasmic reticulum, and is not influenced by inhibitors of slow calcium channels. It was found that trifluoroperazine interferes with calcium transport by sarcoplasmic reticulum in situ, as well as with the role of sarcoplasmic reticulum in contractile activation.     

Protein kinase C activation in human ventricular myocardium

We investigated the effects of 12-deoxyphorbol 13 isobutyrate 20 acetate (DPBA) on contractile function and intracellular calcium handling in normal human ventricular myocardium. The activation of protein kinase C by DPBA resulted in a decrease in sarcoplasmic reticulum calcium release and a reduction in isometric twitch. Force-Calcium relationships were obtained by tetanizing intact muscles or by chemically skinning muscle fibers. These relationships were fitted to a modified Hill function. In intact preparations, DPBA shifted the force-calcium relationship towards higher intracellular calcium concentrations by 0.12 microM (n = 5) and maximal force production was decreased 45.5 +/- 6.1%. These experiments show that protein kinase C activation affects intracellular calcium availability and myofibrillar calcium responsiveness.     

Distribution of calcium during contraction and relaxation of crayfish skeletal muscle fibre.

A model of activation of muscle contraction has been applied to the crayfish isolated skeletal muscle fibre. The model is based on calcium diffusion and binding to specific regulatory sites in a sarcomere. Calcium ions activate interactions of contractile proteins and thus the generation of force. The model quantifies the relation between calcium released from intracellular stores and force elicited. Experimental tension records from isolated crayfish skeletal muscle fibres under voltage clamp conditions are analyzed. Model parameters were determined either via approximation of the onset of tension by the model solution or from the model based relations between the tension maximum, and depolarizing pulse length and amplitude. This allowed to determine time changes of free and bound calcium distribution in the sarcomere and the calcium release from terminal cisternae. The steady state calcium concentration at terminal cisternae showed S-shaped voltage dependence with saturation below approx. 10 mumol/l at positive membrane potentials.     

Mechanisms of stimulated 45Ca efflux in skinned skeletal muscle fibers

Excitation-contraction (E-C) coupling in skeletal muscle can be studied in skinned fibers by direct assay of 45Ca efflux and simultaneous isometric force, under controlled conditions. Recent work provides evidence that such studies can address major current questions about the mechanisms of signal transmission between transverse tubules and sarcoplasmic reticulum and sarcoplasmic reticulum calcium release, as well as operation of the sarcoplasmic reticulum active Ca transport system in situ. Stimulation by imposed ion gradients at constant [K+][Cl-] product results in 45Ca release with two components: a large Ca2+-dependent efflux, responsible for contractile activation, and a small Ca2+-insensitive efflux. The Ca2+-insensitive stimulation is sustained, consistent with sustained depolarization, and appears to gradate the Ca2+-dependent stimulation; this component is likely to reflect intermediate steps in E-C coupling. Several lines of evidence suggest that the depolarizing stimulus acts on the transverse tubules. It is inhibited by the impermeant glycoside ouabain applied before skinning, which should specifically inhibit polarization of subsequently sealed transverse tubules. Sealed polarized transverse tubules also are the only plausible target for stimulation of 45Ca release by monensin and gramicidin D, which can rapidly dissipate Na+ and K+ gradients; a protonophore and the K+-specific ionophore valinomycin are ineffective. Ionophore stimulation is prevented by the permeant glycoside digitoxin; it is also highly Ca2+ dependent. Stimulation of 45Ca release by imposed ion gradients is potentiated by perchlorate, which potentiates charge movements and activation in intact fibers, and is inhibited selectively in highly stretched fibers, presumably by transverse tubule-sarcoplasmic reticulum uncoupling. These results relate the Ca2+-dependent sarcoplasmic reticulum efflux channel to the physiological transverse tubule-sarcoplasmic reticulum coupling pathway, which also could involve Ca2+.     

Doxorubicin induces calcium release from terminal cisternae of skeletal muscle. A study on isolated sarcoplasmic reticulum and chemically skinned fibers

In this study, we investigated the effect of the anticancer drug doxorubicin on Ca2+ fluxes of isolated highly purified sarcoplasmic reticulum fractions (longitudinal tubules and terminal cisternae (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885] and of chemically skinned skeletal muscle fibers of the rabbit. In terminal cisternae, doxorubicin inhibits Ca2+ uptake (IC50 at 0.5 microM) and increases 2.6-fold Ca2+-dependent ATPase rate (half-maximal activation at 3 microM) and unidirectional Ca2+ efflux (8-fold stimulation at 25 microM). On the contrary, doxorubicin is without effect on longitudinal tubules. In skinned muscle fibers, doxorubicin induces rapid and transient Ca2+ release, as measured by tension development (half-maximal stimulation at 6 microM), which is completely and reversibly inhibited by ruthenium red, a known inhibitor of Ca2+ release from isolated terminal cisternae. Doxorubicin has no effect on the sarcoplasmic reticulum Ca2+ pump and on the contractile apparatus of skinned muscle fibers. It is concluded that doxorubicin activates Ca2+ release from sarcoplasmic reticulum and opens a Ca2+ efflux pathway (Ca2+ channel) selectively localized in terminal cisternae. Doxorubicin might interact with Ca2+ channels involved in physiological Ca2+ release.     

Evidence that coated vesicles isolated from brain are calcium- sequestering organelles resembling sarcoplasmic reticulum

Coated vesicles from the brain have been purified to near morphological homogeneity by a modification of the method of Pearse. These vesicles resemble sarcoplasmic reticulum fragments isolated from skeletal muscle. They contain proteins with 100,000- and 55,000-dalton mol wt which co-migrate on polyacrylamide gels, in the presence of sodium dodecyl sulfate, with the two major proteins of the sarcoplasmic reticulum fragment. These vesicles contain adenosine triphosphatase (ATPase) activity which is stimulated by calcium ions in the presence of Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.), displaying maximal activity at 8 x 10(-7) M Ca ++. They take up calcium ions from the medium, and this uptake is stimulated by ATP and by potassium oxalate, a calcium-trapping agent. The 100,000-dalton protein of the coated vesicles displays immunological reactivity with an antiserum directed against the 100,000-dalton, calcium-stimulated ATPase of the sarcoplasmic reticulum. As with the sarcoplasmic reticulum fragment, this protein becomes radiolabeled when coated vesicles are briefly incubated with gamma-labeled [32P]ATP. The possible functions of coated vesicles as calcium-sequestering organelles are discussed.     

Ionized and bound calcium inside isolated sarcoplasmic reticulum of skeletal muscle and its significance in phosphorylation of adenosine triphosphatase by orthophosphate.

Calcium loading of skeletal muscle sarcoplasmic reticulum performed passively by incubation with high calcium concentrations (0.5--15 mM) on ice gives calcium loads of 50--60 nmol/mg sarcoplasmic reticulum protein. This accumulated calcium is not released by EGTA [ethyleneglycol bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid], but almost completely released by ionophore X-537A plus EGTA or phospholipase A plus EGTA treatment and is therefore assumed to be inside the sarcoplasmic reticulum. This calcium is distributed in one saturable and one non-saturable calcium compartment, as derived from the dependence of the calcium load on the calcium concentration in the medium. These compartments are assigned to bound and ionized calcium inside the sarcoplasmic reticulum, respectively. Maximum calcium binding under these conditions was 33 nmol/mg protein with an apparent half-saturation constant of 5,8 nmol/mg free calcium inside, or between 1.2 and 0.6 mM free calcium inside, assuming an average vesicular water space of 5 or 10 microliter/mg protein, respectively. Calcium-dependent phosphorylation of sarcoplasmic reticulum calcium-transport ATPase from orthophosphate depends on the square of free calcium inside, whilst inhibition of phosphorylation depends on the square of free calcium in the medium. Calcium-dependent phosphorylation appears to be determined by the free calcium concentrations inside or outside allowing calcium binding to the ATPase according to the two classes of calcium binding constants for low affinity calcium binding or high affinity calcium binding, respectively. It is further suggested that the saturation of the low-affinity calcium-binding sites of the ATPase facing the inside of the sarcoplasmic reticulum membrane is responsible for the greater apparent orthophosphate and magnesium affinity in calcium-dependent phosphorylation than in calcium-independent phosphorylation from orthophosphate. Maximum calcium-dependent phosphoprotein formation at 20 degrees C and pH 7.0 is about 4 nmol/mg sarcoplasmic reticulum protein.     

Intracellular calcium movements during excitation-contraction coupling in mammalian slow-twitch and fast-twitch muscle fibers

In skeletal muscle fibers, action potentials elicit contractions by releasing calcium ions (Ca(2+)) from the sarcoplasmic reticulum. Experiments on individual mouse muscle fibers micro-injected with a rapidly responding fluorescent Ca(2+) indicator dye reveal that the amount of Ca(2+) released is three- to fourfold larger in fast-twitch fibers than in slow-twitch fibers, and the proportion of the released Ca(2+) that binds to troponin to activate contraction is substantially smaller.     

How perchlorate improves excitation-contraction coupling in skeletal muscle fibers.

The effect of the "chaotropic" anion, perchlorate, on the activation of contraction has been studied in voltage clamped frog skeletal muscle fibers. It was found that the voltage dependence of either the contractile force or the intramembrane charge movement was shifted towards more negative membrane potentials. The maximum values of force or charge movement attained with large depolarizing pulses did not change significantly. It is concluded that a specific perchlorate effect on the movement of charged particles can explain the potentiating effect of perchlorate anions on contractile force, strengthening the view that these charged particles serve as voltage sensors regulating Ca2+ release from the sarcoplasmic reticulum.     

Comparison between strontium and calcium uptake by the fragmented sarcoplasmic reticulum.

The ATP-supported uptake of strontium by the fragmented sarcoplasmic reticulum is monophasic and proceeds more rapidly than the fast uptake of calcium. Strontium uptake is not activated by Pi. The accumulation of strontium is nearly proportional to the external strontium concentration even in the millimolar range. Internal and external strontium quickly equilibrate. One mole of strontium is stored for every mole of ATP split by the Sr2+-activated ATPase. In the absence of oxalate most of the strontium is taken up with a transport ratio of one. On the opposite, the transport ratio of calcium decreases immediately, especially when ADP is not instantaneously phosphorylated to ATP. In this case, energy conversion is uncoupled more effectively by the simultaneous action of ADP and free internal calcium, resulting in the interruption of the fast uptake. After depletion of ATP most of the stored strontium is released and the remaining fraction appears to be not exchangeable. Strontium activates the slow uptake of calcium, but reduces the amplitude of the fast uptake. The calcium induced release of strontium, and vice versa, is partial and transient. The strontium activated ATPase does not transport calcium at low ionic calcium concentrations.     

Inositol trisphosphate and excitation-contraction coupling in skeletal muscle

The role of inositol trisphosphate as a chemical messenger in excitation-contraction coupling is discussed, both in terms of positive and negative results. The evidence presented includes experiments on the effect of inositol trisphosphate in intact and skinned fibers, in calcium release from isolated sarcoplasmic reticulum vesicles, in activation of single calcium release channels incorporated in planar bilayers, and biochemical experiments that have established the presence of all the intermediate steps involved in the metabolism of phosphoinositides, both in intact muscle and in isolated membranes. From these results, it is clear that a role for inositol triphosphate in skeletal muscle function is highly likely; whether this molecule is the physiological messenger in excitation-contraction coupling remains to be established.     

Quantitative determination of the calcium involved in the regulation of the Ca2+-ATPase activity in sarcoplasmic reticulum vesicles

The dependence of the Ca²⁺-ATPase activity of sarcoplasmic reticulum vesicles upon the intravesicular concentration of calcium accumulated after active uptake was studied. The internal calcium concentration was modified by addition of the ionophore A23187 at the steady state of accumulation. About half of the calcium accumulated could be released at low ionophore concentration without any concomitant activation of the Ca²⁺-ATPase. This population of calcium might consist of calcium free in the lumen of the vesicles or bound to the bilayer at sites which do not interact with the ATPase activity. At higher concentrations of ionophore (above 1.75 nmol A23187/mg protein) the release of calcium activated this enzyme. This phenomenon was independent of the extravesicular calcium concentration and might be explained by assuming second species of calcium ions bound to the inner side of the membrane and in close functional interaction with the Ca²⁺-ATPase.     

Effects of prostaglandins and oxytocin on calcium release from a uterine microsomal fraction.

A microsomal fraction resembling striated muscle sarcoplasmic reticulum was isolated from uterine smooth muscle. ATP induces calcium accumulation in this fraction. Increased temperature enhances calcium accumulation and calcium-activated ATPase. In the absence of ATP, approximately 35% of the intrinsic calcium exchanges with the 45Ca in the incubation medium. In the presence of ATP, exchange of intrinsic calcium with 45Ca increases by an amount which equals the ATP-dependent calcium binding. In preparations partially preloaded with calcium, a steady state of bound calcium is reached when the ATP is exhausted. Calcium is released under these conditions by prostaglandins E2 and F2alpha, but not by PGF1beta. The antibiotic ionophores X537A and A23187, as well as oxytocin, also release calcium previously accumulated under ATP stimulation. None of these agents, with the exception of oxytocin, release intrinsic calcium. Thus, the effect of prostaglandins resembles that of the ionophores, suggesting an ionophoretic action of these prostaglandins. The release of calcium conforms with the in vivo smooth muscle contracting action of these agents.     

Ca2+ dependence of tension and ADP production in segments of chemically skinned muscle fibers.

Both ADP production and tension have been measured in segments of chemically skinned fibers contracting at different Ca2+ concentrations. Full mechanical activation occurred between pCa 7.00 and pCa 6.50. The total ATPase was due to both actomyosin and non-actomyosin ATPase. Actomyosin ATPase was observed at pCa 7.09 without accompanying tension. The Ca2+ dependence of tension was steeper than actomyosin ATPase. This finding implies some rate constants of the mechano-chemical cycle are Ca2+ dependent. Non-actomyosin ATPase was measured in fibers stretched beyond overlap of the thick and thin filaments. Sarcoplasmic reticulum was isolated and sarcoplasmic reticulum activity was measured in vitro under the same conditions as the single-fiber experiments. Non-actomyosin ATPase in the single fibers was found to be small compared to maximally activated actomyosin ATPase but larger than the ATPase that could be attributed to sarcoplasmic reticulum activity.     

Effects of fatigue on sarcoplasmic reticulum and myofibrillar properties of rat single muscle fibers.

Force decline during fatigue in skeletal muscle is attributed mainly to progressive alterations of the intracellular milieu. Metabolite changes and the decline in free myoplasmic calcium influence the activation and contractile processes. This study was aimed at evaluating whether fatigue also causes persistent modifications of key myofibrillar and sarcoplasmic reticulum (SR) proteins that contribute to tension reduction. The presence of such modifications was investigated in chemically skinned fibers, a procedure that replaces the fatigued cytoplasm from the muscle fiber with a normal medium. Myofibrillar Ca(2+) sensitivity was reduced in slow-twitch muscle (for example, the pCa value corresponding to 50% of maximum tension was 6.23 +/- 0.03 vs. 5.99 + 0.05, P < 0.01, in rested and fatigued fibers) and not modified in fast-twitch muscle. Phosphorylation of the regulatory myosin light chain isoform increased in fast-twitch muscle. The rate of SR Ca(2+) uptake was increased in slow-twitch muscle fibers (14.2 +/- 1.0 vs. 19.6 +/- 2. 5 nmol. min(-1). mg fiber protein(-1), P < 0.05) and not altered in fast-twitch fibers. No persistent modifications of SR Ca(2+) release properties were found. These results indicate that persistent modifications of myofibrillar and SR properties contribute to fatigue-induced muscle force decline only in slow fibers. These alterations may be either enhanced or counteracted, in vivo, by the metabolic changes that normally occur during fatigue development.     

Aspects of the mechanism of action of local anesthetics on the sarcoplasmic reticulum of skeletal muscle.

1. The effect was studied of local anesthetics (tetracaine, dibucaine, procaine and xylocaine) on the forward and the backward reactions of the calcium pump of skeletal muscle sarcoplasmic reticulum. 2. The inhibition of the rate of calcium uptake, the rate of calcium-dependent ATP splitting and the rate of calcium-dependent ATP-ADP phosphate exchange by sarcoplasmic reticulum in the presence of the above drugs is at least partially due to the inhibition of the phosphoprotein formation from ATP. 3. The rate of the ADP-induced calcium release from sarcoplasmic reticulum and the rate of ATP synthesis driven by the calcium efflux are inhibited on account of a reduction of the phosphoprotein formation by orthophosphate. 4. The phosphorylation of calcium transport ATPase by either ATP or orthophosphate is diminished by the local anesthetics owing to a reduction in the apparent calcium affinity of sarcoplasmic reticulum emmbranes on the outside and on the inside, respectively. 5. The drug-induced calcium efflux from calcium-preloaded sarcoplasmic reticulum vesicles, a reaction not requiring ADP, is probably not mediated by calcium transport ATPase.     

Dual effect of deafferentation on contractile characteristics and sarcoplasmic reticulum properties in rat soleus fibers.

The neural message is known to play a key role in muscle development and function. We analyzed the specific role of the afferent message on the functional regulation of two subcellular muscle components involved in the contractile mechanism: the contractile proteins and the sarcoplasmic reticulum (SR). Rats were submitted to bilateral deafferentation (DEAF group) by section of the dorsal roots L(3) to L(5) after laminectomy. Experiments were carried out in single skinned fibers of the soleus muscle. The maximal force developed by the contractile proteins was increased in the DEAF group compared with control, despite a decrease in muscle mass by 17%. The tension-pCa relationship was shifted toward lower calcium (Ca(2+)) concentrations. Different functional properties of the SR of DEAF soleus were examined by using caffeine-induced contractions. The caffeine sensitivity of the Ca(2+) release was decreased after deafferentation and ryanodine receptor 1 isoform was expressed at a lower level. The rate of Ca(2+) uptake was only slightly increased. The results underlined the dual effect of the afferent input on the functional regulation of both contractile proteins and SR.