Skeletal Sarcoplasmic Reticulum Research Articles

Inhibition of skeletal sarcoplasmic reticulum Ca2+-ATPase activity by deferoxamine nitroxide free radical.

Deferoxamine is an inhibitor of iron-dependent free radical reactions. Despite the antioxidant roles, prolonged clinical use of the chelator is far from benign, and paradoxically, deferoxamine has been shown to promote lipid peroxidation. The possible toxicity of the drug's metabolites, such as deferoxamine nitroxide free radical, deserves attention. We, therefore, tested the hypothesis that deferoxamine nitroxide radicals produced as a result of enzymatic one-electron oxidation of deferoxamine by horseradish peroxidase in the presence of H2O2 are capable of inactivating Ca2+-ATPase of skeletal sarcoplasmic reticulum microsomes as a model system with which to explore the effect of the radical on a biological membrane. Ca2+-ATPase activity of sarcoplasmic reticulum was depressed by exposure to Fenton's reagent (H2O2/FeSO4); the observed effect was significantly enhanced by deferoxamine. We found that the Fenton reaction produced hydroxyl radical, as determined by electron spin resonance spectroscopy. The formation of hydroxyl radical was completely inhibited by deferoxamine; instead, under the same experimental conditions (in the presence of sarcoplasmic reticulum vesicles with or without FeSO4 but without spin trap 5, 5-dimethyl-1-pyrroline N-oxide), the spectral shape and hyperfine coupling constants of electron spin resonance signals confirmed to be long-lived deferoxamine radical were obtained. Furthermore, exposure of sarcoplasmic reticulum vesicles to deferoxamine radical formed by horseradish peroxidase via reaction with H2O2 caused an inhibition of the Ca2+-ATPase activity. The findings show that the sarcoplasmic reticulum vesicles can act as peroxidases and suggest that deferoxamine enhances the decreased Ca2+-ATPase activity afforded by H2O2/FeSO4 due to formation of its metabolites, possibly deferoxamine nitroxide free radical.

Enhanced rotational dynamics of the phosphorylation domain of the Ca-ATPase upon calcium activation.

We have used labeling conditions that permit the specific and covalent attachment of erythrosin isothiocyanate (Er-ITC) to Lys464 within the phosphorylation domain of the Ca-ATPase in skeletal sarcoplasmic reticulum membranes. These labeling conditions do not interfere with high-affinity ATP binding, phosphoenzyme formation, or phosphoenzyme hydrolysis [Huang, S., Negash, S., and Squier, T. C. (1998) Biochemistry 37, 6949-6957]. Thus, we can use frequency-domain phosphorescence spectroscopy to measure the rotational dynamics of the Ca-ATPase stabilized in different enzymatic states corresponding to the absence of bound ligands (E), calcium activation (E x Ca2), the presence of bound nucleotide (E x ATP), and formation of phosphoenzyme (E-P). We resolve three rotational correlation times corresponding to (i) a large-amplitude domain motion of the phosphorylation domain (phi1 approximately 5 +/- 1 micros), (ii) overall protein rotational motion with respect to the membrane normal (phi2 approximately 50 +/- 10 micros), and (iii) the rotational motion of the SR vesicles (phi3 approximately 1.1 +/- 0.4 ms). No differences are observed in the rotational dynamics of E, E x ATP, or E-P, indicating that phosphoenzyme formation or nucleotide binding result in no global structural changes involving the phosphorylation domain. In contrast, calcium activation enhances the amplitude of motion of the phosphorylation domain. These observed calcium-dependent changes in rotational dynamics result from structural changes within a single Ca-ATPase polypeptide chain, since protein-protein interactions do not change upon calcium binding. Thus, calcium binding induces concerted domain motions within a single Ca-ATPase polypeptide chain that may play a critical role in facilitating substrate binding and utilization.

9-Methyl-7-bromoeudistomin D induces Ca 2+ release from cardiac sarcoplasmic reticulum

9-Methyl-7-bromoeudistomin D (MBED), the most powerful caffeine-like releaser of Ca 2+ from skeletal muscle sarcoplasmic reticulum, induced Ca 2+ release from the cardiac sarcoplasmic reticulum. MBED (5 μM) and caffeine (1 mM) caused rapid Ca 2+ release from the fragmented cardiac sarcoplasmic reticulum in a Ca 2+ electrode experiment. [ 3 H ]MBED bound to a single class of high-affinity binding sites in cardiac sarcoplasmic reticulum membranes ( K d=150 nM). These results suggest that MBED binds to a specific binding site on cardiac sarcoplasmic reticulum membranes to induce Ca 2+ release from the cardiac sarcoplasmic reticulum. Thus, MBED is a useful probe for characterizing Ca 2+ release the channels not only in skeletal sarcoplasmic reticulum but also in cardiac sarcoplasmic reticulum.

Polylysine-induced rapid Ca2+ release from cardiac sarcoplasmic reticulum.

The rapid kinetics of polylysine-induced Ca2+ release from cardiac sarcoplasmic reticulum (SR) was assessed in combination with its effect on ryanodine binding. SR vesicles were isolated from canine cardiac SR. The time course of SR Ca2+ release was continuously monitored by a stopped-flow apparatus, and [3H]ryanodine binding was done by using the filtration method. The initial rate of polylysine-induced Ca2+ release from cardiac SR revealed different concentration dependence from those observed in skeletal SR. The initial rate peaked at 0.11 microM, followed by a decrease at higher concentrations in skeletal SR, whereas it increased to 3.7 microM in cardiac SR. The [3H]ryanodine binding was also stimulated by polylysine with an identical parallelism with Ca2+ release in terms of polylysine concentration dependence. Thus we demonstrated that the cardiac SR Ca2+ release channel is sensitive to activation by polylysine and that there is a difference in the concentration dependence of polylysine-induced activation of cardiac and skeletal SR Ca2+ release channels.

Erythrosin isothiocyanate selectively labels lysine464 within an ATP-protectable binding site on the Ca-ATPase in skeletal sarcoplasmic reticulum membranes.

Conditions that permit the selective modification of an ATP-protectable site on the Ca-ATPase in skeletal sarcoplasmic reticulum (SR) membranes using erythrosin isothiocyanate (Er-ITC) have been identified. The major labeling site for Er-ITC has been identified using reversed-phase HPLC and positive FAB mass spectrometry after exhaustive tryptic digestion of the Er-ITC-modified Ca-ATPase. An ATP-protectable peptide corresponding to M452NVFNTEVRNLSK464VER467 is modified by Er-ITC, the average mass of which is 2830.1 +/- 0.3 Da. The exclusive modification of lysine residues indicates Lys464 as the site of Er-ITC modification. Derivatization with Er-ITC diminishes the secondary activation of steady-state ATPase activity and the rate of dephosphorylation by millimolar concentrations of ATP. In contrast, in the presence of micromolar ATP concentrations Er-ITC modification of the Ca-ATPase does not affect (i) the apparent affinity of ATP, (ii) the maximal extent of phosphoenzyme formation by ATP, (iii) the rate of steady-state ATP hydrolysis, or (iv) the rate of dephosphorylation of the Ca-ATPase. Furthermore, ATP utilization by the Ca-ATPase is unaffected by detergent solubilization, irrespective of Er-ITC modification, indicating that the secondary activation of ATP hydrolysis involves a single Ca-ATPase polypeptide chain. Therefore, Er-ITC does not interfere with the normal structural transitions associated with phosphoenzyme decay. Rather, these results indicate that Er-ITC bound to Lys464 interferes with either ATP binding to a low-affinity site or the associated structural transitions that modulate the rate of enzyme dephosphorylation.

Fluorescent Probing with Felodipine of the Dihydropyridine Receptor and Its Interaction with the Ryanodine Receptor Calcium Release Channel

The intensity of the fluorescence emission of the fluorescent 1,4-dihydropyridine (DHP) derivative felodipine increased upon binding to both isolated cardiac sarcolemma (SLM) and skeletal muscle sarcoplasmic reticulum (SR) preparations, the latter containing SR-transversal tubule junctional diads and triads. The fluorescence enhancement was due to the binding of felodipine to high-affinity (Kd's of 0.35 and 1.25 nM in cardiac SLM and skeletal SR, respectively) 1,4-dihydropyridine sites of the dihydropyridine receptor (DHPR), as evidenced in competition experiments with the DHP analog isradipine. In both cardiac SLM and SR, the felodipine fluiorescence was sensitive to conformational changes of the DHPR, as diltiazem that binds to DHPR at a separate site altered the values of both the Kdand the Hill coefficient characteristic for felodipine binding. In skeletal muscle membranes containing intact TT-SR junctions, ryanodine, a specific ligand of the ryanodine receptor calcium release channel (RyRC), also induced changes in felodipine fluorescence, which was eliminated by detergent and high-salt treatment to solubilize the RyRC. These results suggest that i) felodipine fluorescence is useful to probe conformational changes of the DHPR and ii) coupled conformational changes between the DHPR and the RyRC in skeletal muscle indeed occur and could be monitored by measuring felodipine fluorescence.

Differential effects of general anesthetics on the quaternary structure of the Ca-ATPases of cardiac and skeletal sarcoplasmic reticulum.

The effects of the general anesthetics hexanol, halothane, and diethyl ether on Ca-ATPase activity and on the oligomeric state of the Ca-ATPase of sarcoplasmic reticulum (SR) from cardiac and skeletal muscle were investigated. The effects of these general anesthetics on Ca-ATPase activity were similar in cardiac and skeletal SR and were characterized by stimulation of Ca-ATPase activity at lower concentrations of anesthetics and inhibition at higher concentrations. The distribution of the Ca-ATPase among its oligomeric states was estimated from the time-resolved phosphorescence anisotropy (TPA) decay of SR in which Ca-ATPase was covalently labeled with erythrosin isothiocyanate (ERITC) or with erythrosin iodoacetamide (ERIA). In contrast to the similar responses of Ca-ATPase activity, there were marked differences in the responses to general anesthetics of the TPA decay between cardiac and skeletal SR. In cardiac SR hexanol, halothane, and diethyl ether caused pronounced increases in the limiting anisotropy at very long times (r infinity), which indicate increases in the fraction of oligomers too large to rotate on the millisecond time scale of the experiments. In skeletal SR, by contrast, there were no significant changes in r infinity in response to the three general anesthetics. This difference between cardiac and skeletal SR in response to general anesthetics is not due to the presence of phospholamban in cardiac SR, since SR from AT-1 cells, which have the SERCA2a isoform of Ca-ATPase, but only trace levels of phospholamban, have increases in r infinity in response to the general anesthetics that resemble those in cardiac SR. Experiments with cardiac SR labeled with ERIA give similar results, showing that the results with ERITC are not an artifact of the labeling procedure. Increasing the ionic strength with LiCl diminished the proportion of large immobile oligomers of cardiac Ca-ATPase under control conditions but enhanced the formation of large oligomers in response to hexanol.

Hypochlorous acid inhibits Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum.

Hypochlorous acid (HOCl) is produced by polymorphonuclear leukocytes that migrate and adhere to endothelial cells as part of the inflammatory response to tissue injury. HOCl is an extremely toxic oxidant that can react with a variety of cellular components, and concentrations reaching 200 microM have been reported in some tissues. In this study, we show that HOCl interacts with the skeletal sarcoplasmic reticulum Ca(2+)-adenosinetriphosphatase (ATPase), inhibiting transport function, HOCl inhibits sarcoplasmic reticulum Ca(2+)-ATPase activity in a concentration-dependent manner with a concentration required to inhibit ATPase activity by 50% of 170 microM and with complete inhibition of activity at 3 mM. A concomitant reduction in free sulfhydryl groups after HOCl treatment was observed, paralleling the inhibition of ATPase activity. It was also observed that HOCl inhibited the binding of the fluorescent probe fluorescein isothiocyanate to the ATPase protein, indicating some structural damage may have occurred. These findings suggest that the reactive oxygen species HOCl inhibits ATPase activity via a modification of sulfhydryl groups on the protein, supporting the contention that reactive oxygen species disrupt the normal Ca(2+)-handling kinetics in muscle cells.

Conducting and voltage-dependent behaviors of the native and purified SR Ca 2+-release channels from the canine diaphragm

The ryanodine-sensitive Ca 2+-release channel of the canine diaphragm sarcoplasmic reticulum (SR) was characterized using biochemical assays and the planar lipid bilayer technique. Diaphragm SR membranes have a [ 3 H ]ryanodine-binding capacity ( B max) of 1.2 pmol/mg protein and a binding affinity ( K D) of 6.3 nM. The conductance of the native channel was 330 pS in 50 mM/250 mM trans/ cis CsCH 3SO 3 and was reduced to 71 pS by 10 mM Ca 2+ trans. The Ca 2+-release channel was purified as a 400 kDa protein on SDS-PAGE and displayed a conductance of 715 pS in 200 mM KCl. The native and purified Ca 2+ channels were activated by micromolar Ca 2+ and ATP and inhibited by Mg 2+, ryanodine and ruthenium red. Although diaphragm muscle contraction was shown to depend on extracellular Ca 2+ like cardiac muscles, we provide evidence that the diaphragm SR Ca 2+-release channel may be classified as a skeletal ryanodine receptor isoform. First, the IC 50 for [ 3 H ]ryanodine binding was in the same range as estimated for skeletal SR, with 20 nM. Second, the channel was maximally activated by 10–30 μM cytoplasmic Ca 2+ and inhibited at higher concentrations. Third, ryanodine binding to the diaphragm SR was less sensitive to Ca 2+ than cardiac SR, with EC 50 values of 50 and 1 μM, respectively. Finally, Ca 2+-release activity and [ 3 H ]ryanodine binding capacity of the diaphragm and skeletal SR were similarly more sensitive to Mg 2+ than cardiac SR. Together, these results suggest a predominantly skeletal-type of excitation-contraction coupling in the diaphragm.

Preservation of the native structure and function of Ca2+-ATPase from sarcoplasmic reticulum: solubilization and reconstitution by new short-chain phospholipid detergent 1,2-diheptanoyl-sn-phosphatidylcholine.

The properties of Ca2+-ATPase purified and reconstituted from rabbit skeletal sarcoplasmic reticulum (SR) has been studied in comparison with the preparations obtained by the commonly used detergent poly(oxyethylene)8-lauryl ether (C12E8) and the bile salt detergents cholate and deoxycholate. 1,2-Diheptanoyl-sn-phosphatidylcholine (DHPC) has been shown to be excellent for solubilizing a wide variety of membrane proteins [Kessi, Poiree, Wehrli, Bachofen, Semenza and Hauser (1994) Biochemistry 33, 10825-10836]. The DHPC method consistently gave higher yields of purified Ca2+-ATPase with a greater specific activity than the methods with C12E8, cholate, or deoxycholate. DHPC and C12E8 were superior to cholate and deoxycholate in active enzyme yields and specific activity. DHPC-solubilized Ca2+-ATPase purified on a density gradient retained the E1Ca-E1(*)Ca conformational transition, whereas the enzyme from the C12E8 purification did not retain this transition. The coupling of Ca2+ transported to ATP hydrolysed in the DHPC-purified enzyme was maximal and matched the values obtained with native SR, whereas the coupling was much lower for the C12E8-purified enzyme. The specific activity of Ca2+-ATPase reconstituted into dioleoylphosphatidylcholine vesicles with DHPC was up to 2-fold greater than that achieved with C12E8, and is comparable to that measured in the native SR. Finally, the dissociation of Ca2+-ATPase into monomers by DHPC preserved the ATPase activity, whereas similar dissociation by C12E8 gave only one-sixth the activity of that obtained with DHPC. These studies show that the Ca2+-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C12E8 in significant ways, making it a preparation suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of membrane proteins.

A carboxy-terminal peptide of the alpha 1-subunit of the dihydropyridine receptor inhibits Ca(2+)-release channels.

Excitation-contraction coupling in skeletal muscle is thought to involve a physical interaction between the alpha 1-subunit of the dihydropyridine receptor (DHPR) and the sarcoplasmic reticulum (SR) Ca(2+)-release channel (also known as the ryanodine receptor). Considerable evidence has accumulated to suggest that the cytoplasmic loop between domains II and III of the DHPR alpha 1-subunit is at least partially responsible for this interaction. Other parts of this subunit or other subunits may, however, contribute to the functional and/or structural coupling between these two proteins. A synthetic peptide corresponding to a conserved sequence located between amino acids 1487 and 1506 in the carboxy terminus of the alpha 1-subunit inhibits both [3H]ryanodine binding to skeletal and cardiac SR membranes and the activity of skeletal SR Ca(2+)-release channels reconstituted into planar lipid bilayers. A second, multiantigenic peptide synthesized to correspond to the same sequence inhibits both binding and channel activity at lower concentrations than the linear peptide. These peptides slow the rate at which [3H]ryanodine binds to its high-affinity binding site and decrease the rate at which [3H]ryanodine dissociates from this site. A third polypeptide synthesized in Escherichia coli and corresponding to amino acids 1381-1627 and encompassing the above sequence has similar effects. This portion of the alpha 1-subunit of the transverse tubule DHPR is therefore a candidate for contributing to the interaction of this protein with the Ca(2+)-release channel.

Noncoplanar PCB 95 alters microsomal calcium transport by an immunophilin FKBP12-dependent mechanism.

Ortho-substituted polychlorinated biphenyls (PCBs) have been shown to alter microsomal Ca2+ transport by selective interaction with ryanodine receptors (RyRs) of muscle sarcoplasmic reticulum (SR) and brain endoplasmic reticulum. The mechanism underlying the actions of PCBs on Ca2+ transport is further elucidated with skeletal SR enriched in Ry1R. Disruption of the association between immunophilin FKBP12 and Ry1R with FK 506 or rapamycin completely eliminates PCB 95-enhanced binding of [3H]ryanodine (IC50 approximately 35 microM) to Ry1R and PCB 95-induced release of Ca2+ from actively loaded SR vesicles (IC50 approximately 11 microM), demonstrating a FKBP12-dependent mechanism. FK 506 selectively eliminates PCB 95-induced Ca2+ release from SR because Ry1R maintains responsiveness to caffeine and Ca2+. PCB 95 and FK 506 are used to examine the relationship between ryanodine-sensitive Ca2+ channels and ryanodine-insensitive Ca2+ leak pathways present in SR vesicles. Micromolar ryanodine completely blocks ryanodine-sensitive Ca2+ efflux but neither eliminates the ryanodine-insensitive Ca2+ leak unmasked by thapsigargin nor enhances the loading capacity of SR vesicles. PCB 95 alone enhances thapsigargin evoked Ca2+ release and therefore diminishes the loading capacity of SR vesicles. However, in the presence of micromolar ryanodine, PCB 95 dose-dependently eliminates the Ca2+ leak unmasked by thapsigargin and significantly enhances the loading capacity of SR vesicles. The actions of PCB 95 on SR-loading capacity are additive with those of FK 506. Structural specificity for these novel actions are further demonstrated with coplanar PCB 126, which is inactive toward Ry1R and lacks the ability to alter the Ca2+ leak pathway. The results reveal that FKBP12 relates ryanodine-insensitive Ca2+ "leak" and ryanodine-sensitive Ca2+ channel efflux pathways of SR by modulating distinct conformations Ry1R complexes. Noncoplanar PCBs, like PCB 95, alter SR Ca2+ buffering by an FKBP12-mediated mechanism. An immunophilin-based mechanism could account for the toxic actions attributed to certain noncoplanar PCB congeners.

Bastadins relate ryanodine-sensitive and -insensitive Ca2+ efflux pathways in skeletal SR and BC3H1 cells.

Bastadins potently interact with the FK-506-binding protein of 12 kDa (FKBP12)-ryanodine receptor (Ry1R) complex in skeletal muscle to enhance a high-affinity ryanodine binding conformation (M. M. Mack, T. F. Molinski, E. D. Buck, and I. N. Pessah. J. Biol. Chem. 269: 23236-23249, 1994). Bastadins are used to examine the relationship between ryanodine-sensitive and ryanodine-insensitive Ca2+ efflux pathways that coexist in junctional sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle and differentiated BC3H1 cells. Complete block of caffeine-sensitive Ca2+ channels with micromolar ryanodine or ruthenium red does not alter the steady-state loading capacity of SR. Inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps with thapsigargin unmasks a ryanodine- and ruthenium red-insensitive Ca2+ efflux pathway. Bastadin 5 alone does not inhibit Ca2+ efflux unmasked by inhibition of SERCA pumps, but, in combination with blocking concentrations of ryanodine or ruthenium red, it eliminates the ryanodine-insensitive Ca2+ "leak" and enhances steady-state loading capacity of SR vesicles approximately 2.5-fold. These actions of bastadins occur in the same concentration range that enhances the number of high-affinity binding sites for [3H]ryanodine (50% effective concentration of approximately 2 microM). Similar effects on SR Ca2+ transport are found with FK-506 and ryanodine in combination. Block of Ry1R in intact BC3H1 cells with ryanodine does not eliminate the prominent Ca2+ leak unmasked by thapsigargin. A membrane-permeant mixture of bastadins in combination with ryanodine nearly eliminates the Ca2+ leak unmasked by thapsigargin, even though the Ca2+ stores are replete. The requirement of both a known Ry1R blocker and bastadins in combination provides a pharmacological link between ryanodine-sensitive Ca2+ channels and ryanodine-insensitive leak pathways in isolated junctional SR and BC3H1 cells. Together, these results strongly suggest that bastadins, through their modulatory actions on the FKBP12-Ry1R complex, convert ryanodine-insensitive leak states into ryanodine-sensitive channels that recognize [3H]ryanodine with high affinity.

Properties of ryanodine receptors in cultured cerebellar granule neurons: effects of hexachlorocyclohexane isomers and calcium.

The binding of [3H]ryanodine was determined in microsomal membrane preparations obtained from cultured cerebellar granule cells. A KD of 1 nM and a Bmax of 64 fmol/mg protein were calculated from saturation experiments. This binding was calcium dependent and maximum values were obtained at 100-300 microM Ca+2. Caffeine increased [3H]ryanodine binding only at Ca+2 concentrations lower than optimum. The binding of [3H]ryanodine was inhibited by ruthenium red, procaine and the delta-isomer of hexachlorocyclohexane (delta-HCH). Dantrolene, a ryanodine receptor antagonist in skeletal sarcoplasmic reticulum, and the pesticide gamma-HCH (lindane) had no effect on [3H]ryanodine binding. The obtained binding parameters, the Ca+2 dependence and the effects of the agents tested agree with previous reports using brain microsomal membranes, further indicating a neuronal localization of [3H]ryanodine binding sites. When the interaction between dantrolene and gamma- and delta-HCH was tested, no changes were detected on the effects of HCH isomers on [3H]ryanodine binding. Dantrolene, which inhibits Ca+2 release from sarcoplasmic reticulum and from unidentified internal Ca+2 stores in neurons, also inhibits the intracellular Ca+2 mobilization induced by gamma-HCH but only marginally that induced by delta-HCH in the same preparation of cerebellar granule cells (Rosa et al.; Toxicol Appl Pharmacol, in press). Thus, the results obtained in this work verify the presence of different intracellular sites of action for the two HCH isomers: the ryanodine Ca+2 channel for delta-HCH and an unidentified dantrolene-sensitive Ca+2 channel for the gamma-HCH isomer.

Effect of dantrolene sodium on calcium-overloaded heart.

Spontaneous asynchronous contractile activity caused by spontaneous release of calcium ions (Ca2+) from the sarcoplasmic reticulum (SR) is thought to be the cause of deterioration of ventricular function under conditions of calcium overload. We examined whether dantrolene sodium, which can inhibit Ca2+ release from the skeletal SR, improves the systolic and diastolic function of calcium-overloaded hearts. In isolated hamster left ventricles, the concentration of Ca2+ in the perfusate ([Ca2+]o) was increased from 1 mmol/L to 7 mmol/L in 1-mmol/L steps in the absence (control, n = 6) and presence of dantrolene sodium (11.8 mumol/L, n = 5). Left ventricular developed pressure and its maximum rate of rise (max dP/dt) increased with an increase in [Ca2+]o up to 4 mmol/L, and decreased with a further increase in [Ca2+]o. In the presence of dantrolene sodium, developed pressure and max dP/dt increased up to 5 mmol/L [Ca2+]o. Thus, dantrolene sodium improves Ca2+ tolerance. In isolated ventricles perfused with 1 mmol/L [Ca2+]o, dantrolene sodium decreased developed pressure by 33.7 +/- 7.4% and max dP/dt by 37.4 +/- 5.6% (mean +/- SEM, n = 8) at 1 mmol/L [Ca2+]o. In contrast, at 5 mmol/L [Ca2+]o ('calcium-overloaded state'), dantrolene sodium increased developed pressure by 6.8 +/- 2.6% and max dP/dt by 14.4 +/- 5.7%, and decreased the end-diastolic pressure by 5.3 +/- 1.9% (n = 8). Dantrolene sodium partially suppressed the spontaneous contractile activities observed microscopically on the epicardium of ventricles perfused with 5 mmol/L [Ca2+]o. Dantrolene sodium improved the Ca2+ tolerance of left ventricles and exerted positive inotropic effects and decreased diastolic stiffness in calcium-overloaded hamster left ventricles by suppressing spontaneous contractile activity.

A new scorpion polypeptide enhances the binding of radiolabeled-ryanodine on ryanodine receptor in sarcoplasmic reticulum of rabbit skeletal muscle

RYANODINE is a toxic alkaloid from the plant Ryania speciosa Vahl. It selectively binds to the Ca~(2+) release channel on skeletal and cardiac heavy sarcoplasmic reticulum (HSR) and has been...

Nitric oxide activates skeletal and cardiac ryanodine receptors

The endothelial-derived relaxing factor, nitric oxide (NO ·) has been shown to depress force in smooth and cardiac muscles through the activation of guanylyl cyclase and an increase in cGMP. In fast skeletal muscle, NO (i.e. NO-related compounds) elicits a modest decrease in developed force, but in contracting muscles NO increases force by a mechanism independent of cGMP. We now demonstrate an alternative mechanism whereby NO triggers Ca 2+ release from skeletal and cardiac sarcoplasmic reticulum (SR). NO delivered in the form of NO gas, NONOates (a class of sulfur-free compounds capable of releasing NO), or S-nitrosothiols (R-SNO) oxidized or transnitrosylated regulatory thiols on the release channel (or ryanodine receptor, RyR), resulting in channel opening and Ca 2+ release from skeletal and cardiac SR. The process was reversed by sulfhydryl reducing agents which promoted channel closure and Ca 2+ reuptake by ATP-driven Ca 2+ pumps. NO did not directly alter Ca 2+-ATPase activity but increased the open probability of RyRs reconstituted in planar bilayers and inhibited [ 3H]-ryanodine binding to RyRs. The formation of peroxynitrite or thiyl radicals did not account for the reversible R-SNO-dependent activation of RyRs. Ca 2+ release induced by nitric oxide free radicals (NO ·) was potentiated by cysteine providing compelling evidence that NO · in the presence of O 2 formed nitrosylated cysteine followed by the transnitrosation of regulatory thiols on the RyR to activate the channel. These findings demonstrate direct interactions of NO derivatives with RyRs and a new fundamental mechanism to regulate force in striated muscle.

Volatile anaesthetic effects on calcium conductance of planar lipid bilayers formed with synthetic lipids or extracted lipids from sarcoplasmic reticulum

Volatile anaesthetics are known to increase leakage of calcium from the light fraction of skeletal sarcoplasmic reticulum (L-SR) which has no calcium release channels. To explore the role of the lipid environment, we have examined the effect of volatile anaesthetics on calcium conductance (gCa) of lipid membranes. Planar lipid bilayers were formed with a mixture of synthetic phospholipids and cholesterol, resembling the composition of SR membranes, or with lipids extracted from skeletal L-SR, gCa was estimated by calculating the calcium transference number (tCa) using diffusion potential measurements. Membranes formed with L-SR-extracted lipids had a higher gCa than membranes formed with synthetic lipids. Volatile anaesthetics increased total conductance and gCa in a dose-dependent manner, but did not affect tCa or membrane specific capacitance. In membranes formed with L-SR-extracted lipids, isoflurane induced the largest increase in gCa (1260 (SEM 304) % increase, n = 4, 0.94 mmol litre-1), followed by enflurane (264 (75)%, n = 5, 1.88 mmol litre-1) and halothane (53 (33)%, n = 5; 1.54 mmol litre-1). In membranes formed with synthetic lipids, volatile anaesthetic-induced increases in gCa followed the same trend but were larger. Volatile anaesthetics increased gCa without changing the ionic selectivity of membranes. However, the magnitude of the increase in gCa in the presence of volatile anaesthetics cannot account for the previously observed calcium leakage from L-SR vesicles. Therefore, the volatile anaesthetic-induced increase in calcium leakage in L-SR vesicles must be mediated via other pathways involving membrane proteins.

Ptilomycalin A, a novel Na +,K +- or Ca 2+-ATPase inhibitor, competitively interacts with ATP at its binding site

Ptilomycalin A inhibited the brain Na +,K +-ATPase and Ca 2+-ATPase from skeletal sarcoplasmic reticulum with an IC 50 value of 2 μM and 10 μM, respectively. Kinetic analysis of the inhibitory effects of ptilomycalin A suggests that the inhibition of Na +,K +-ATPase is a competitive-, an uncompetitive- and an anticompetitive-type with respect to ATP, Na + and K +, respectively. The inhibition of Ca 2+-ATPase by ptilomycalin A is a competitive- or an uncompetitive-type with respect to ATP or Ca 2+, respectively. These results suggest that ptilomycalin A interacts with ATP at the ATP binding site of Na +,K +-ATPase or Ca 2+-ATPase. Ptilomycalin A has become a useful biochemical tool for clarifying the ATP binding site in both enzymes.

4-Chloro-m-cresol: A specific tool to distinguish between malignant hyperthermia-susceptible and normal muscle

Single-channel recordings have indicated that ryanodine receptor (RyR1) mutation Arg615Cys of porcine malignant hyperthermia-susceptible (MHS) muscle is not directly associated with the enhanced caffeine sensitivity of MH(S) muscle [1]. In the present study, the effect of a novel activator of RyR1, 4-chloro-m-cresol (4-CmC), was investigated on high-affinity [ 3H]ryanodine binding to porcine skeletal sarcoplasmic reticulum. The 4-CmC affinity of [ 3H]ryanodine binding to MHS vesicles was 2-fold higher compared to that in normal tissue. This enhanced affinity was confirmed when the effect of 4-CmC on [ 3H]ryanodine binding to the isolated CHAPS-solubilized MHS RyR1 was investigated. 4-CmC is, therefore, suggested to be a potent tool to distinguish between Ca 2+ release from MHS and normal muscle.