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.     

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.     

Functional characterization of junctional terminal cisternae from mammalian fast skeletal muscle sarcoplasmic reticulum

Junctional terminal cisternae are a recently isolated sarcoplasmic reticulum fraction containing two types of membranes, the junctional face membrane with morphologically intact "feet" structures and the calcium pump membrane [Saito, A., Seiler, S., Chu, A., & Fleischer, S. (1984) J. Cell Biol. 99, 875-885]. In this study, the Ca2+ fluxes of junctional terminal cisternae are characterized and compared with three other well-defined fractions derived from the sarcotubular system of fast-twitch skeletal muscle, including light and heavy sarcoplasmic reticulum, corresponding to longitudinal and terminal cisternae regions of the sarcoplasmic reticulum, and isolated triads. Functionally, junctional terminal cisternae have low net energized Ca2+ transport measured in the presence or absence of a Ca2+-trapping anion, as compared to light and heavy sarcoplasmic reticulum and triads. Ca2+ transport and Ca2+ pumping efficiency can be restored to values similar to those of light sarcoplasmic reticulum with ruthenium red or high [Mg2+]. In contrast to junctional terminal cisternae, heavy sarcoplasmic reticulum and triads have higher Ca2+ transport and are stimulated less by ruthenium red. Heavy sarcoplasmic reticulum appears to be derived from the nonjunctional portion of the terminal cisternae. Our studies indicate that the decreased Ca2+ transport is referable to the enhanced permeability to Ca2+, reflecting the predominant localization of Ca2+ release channels in junctional terminal cisternae. This conclusion is based on the following observations: The Ca2+, -Mg2+ -dependent ATPase activity of junctional terminal cisternae in the presence of a Ca2+ ionophore is comparable to that of light sarcoplasmic reticulum when normalized for the calcium pump protein content; i.e., the enhanced Ca2+ transport cannot be explained by a faster turnover of the pump. Ruthenium red or elevated [Mg2+] enhances energized Ca2+ transport and Ca2+ pumping efficiency in junctional terminal cisternae so that values approaching those of light sarcoplasmic reticulum are obtained. Rapid Ca2+ efflux in junctional terminal cisternae can be directly measured and is blocked by ruthenium red or high [Mg2+]. Ryanodine at pharmacologically significant concentrations blocks the ruthenium red stimulation of Ca2+ loading. Ryanodine binding in junctional terminal cisternae, which appears to titrate Ca2+ release channels, is 2 orders of magnitude lower than the concentration of the calcium pump protein. By contrast, light sarcoplasmic reticulum has a high Ca2+ loading rate and slow Ca2+ efflux that are not modulated by ruthenium red, ryanodine, or Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid filtration studies of Ca2+-induced Ca2+ release from skeletal sarcoplasmic reticulum. Role of monovalent ions

We have developed a rapid filtration technique for the measurement of Ca2+ release from isolated sarcoplasmic reticulum vesicles. Using this technique, we have studied the Ca2+-induced Ca2+ release of sarcoplasmic reticulum vesicles from rabbit skeletal muscle passively loaded with 5 mM Ca2+. The effect of known effectors (adenine nucleotides and caffeine) and inhibitors (Mg2+ and ruthenium red) of this release were investigated. In a medium composed of 100 mM KCl buffered at pH 6.8 with 20 mM K/3-(N-morpholino)propanesulfonic acid the Ca2+ release rate was maximal (500 nmol of Ca2+ released.(mg of protein)-1.s-1) at 1 micron external Ca2+ and 5 mM ATP. We also observed a rapid Ca2+ release induced by micromolar Ag+ in the presence of ATP (at 1 nM Ca2+). The Ag+-induced Ca2+ release was totally inhibited by 5 micron ruthenium red. We have also investigated the effect of monovalent ions on the Ca2+ release elicited by Ca2+ or Ag+. We show that the Ca2+ release rate: 1) was dependent upon the presence of K+ or Na+ in the release medium and 2) was influenced by a K+ gradient created across the sarcoplasmic reticulum membrane. These results directly support the idea of the involvement of an influx of K+ (through K+ channels) during the Ca2+ release and allow to reconsider a possible influence of the membrane potential of the sarcoplasmic reticulum on the Ca2+ release.     

Optical probe responses on sarcoplasmic reticulum: oxacarbocyanines as probes of membrane potential.

The relationship between Ca2+ fluxes and the ion diffusion potential was analyzed on sarcoplasmic reticulum membranes using oxacarbocyanine dyes as optical probes for membrane potential. 3.3'-Diethyloxodicarbocyanine responds to ATP-induced Ca2+ uptake by isolated sarcoplasmic reticulum vesicles with a decrease in absorbance at 600 nm. The optical change is reversed during Ca2+ release from sarcoplasmic reticulum induced by KCl or by ADP and inorganic phosphate. The absorbance changes are largely attributable to the binding of accumulated Ca2+ to the membrane. There is no indication that sustained changes in membrane diffusion potential would accompany pump-mediated Ca2+ fluxes. A large change in the absorbance of 3,3'-diethyloxodicarbocyanine was observed on sarcoplasmic reticulum vesicles under the influence of membrane potential generated by valinomycin in the presence of a K+ gradient or by ionophore A23187 in the presence of a Ca2+ gradient. The maximum of the potential-dependent absorbance change is at 575--580 nm. The potentials generated by valinomycin or ionophore A23187 are short-lived due to the high permeability of sarcoplasmic reticulum membranes for cations and anions. There is no correlation between the direction and magnitude of the artifically imposed membrane potential and the rate of Ca2+ uptake or release by isolated sarcoplasmic reticulum vesicles.     

The effect of ionomycin on calcium fluxes in sarcoplasmic reticulum vesicles and liposomes.

Ionomycin, a recently discovered calcium ionophore, inhibits the ATP-dependent active Ca2+ transport of rabbit sarcoplasmic reticulum vesicles at concentrations as low as 10(-8) to 10(-6) M. The effect is due to an increase in the Ca2+ permeability of the membrane which is also observed on liposomes. The inhibition of Ca2+ uptake is accompanied by an increase in the Ca2+-sensitive ATPase activity of sarcoplasmic reticulum vesicles.     

Altered stored calcium release in skeletal myotubes deficient of triadin and junctin

Triadin and junctin are integral sarcoplasmic reticulum membrane proteins that form a macromolecular complex with the skeletal muscle ryanodine receptor (RyR1) but their roles in skeletal muscle calcium homeostasis remain incompletely understood. Here we report that delivery of siRNAs specific for triadin or junctin into C2C12 skeletal myoblasts reduced the expression of triadin and junctin in 8-day-old myotubes by 80 and 100%, respectively. Knocking down either triadin or junctin in these cells reduced Ca2+ release induced by depolarization (10mM KCl) by 20-25%. Unlike triadin knockdown myotubes, junctin knockdown and junctin/triadin double knockdown myotubes also had reduced Ca2+ release induced by 400 microM 4-chloro-m-cresol, 10mM caffeine, 400 microM UTP, or 1 microM thapsigargin. Thus, knocking down junctin compromised the Ca2+ stores in the sarcoplasmic reticulum of these cells. Our subsequent studies showed that in junctin knockdown myotubes at least two sarcoplasmic reticulum proteins (RyR1 and skeletal muscle calsequestrin) were down-regulated while these proteins' mRNA expression was not affected. The results suggest that triadin has a role in facilitating KCl depolarization-induced Ca2+ release in contrast to junctin which has a role in maintaining sarcoplasmic reticulum Ca2+ store size in C2C12 myotubes.     

Mechanism of the stimulation of Ca2+-dependent ATPase of skeletal muscle sarcoplasmic reticulum by protein kinase

Sarcoplasmic reticulum isolated from moderately fast rabbit skeletal muscle contains intrinsic adenosine 3',5'-monophosphate (cAMP)-independent protein kinase activity and a substrate of 100 000 Mr. Phosphorylation of skeletal sarcoplasmic reticulum by either endogenous membrane bound or exogenous cAMP-dependent protein kinase results in stimulation of the initial rates of Ca2+ transport and Ca2+-ATPase activity. To determine the molecular mechanism by which protein kinase-dependent phosphorylation regulates the calcium pump in skeletal sarcoplasmic reticulum, we examined the effects of protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Skeletal sarcoplasmic reticulum vesicles were preincubated with cAMP and cAMP-dependent protein kinase in the presence (phosphorylated sarcoplasmic reticulum) and absence (control sarcoplasmic reticulum) of adenosine 5'-triphosphate (ATP). Control and phosphorylated sarcoplasmic reticulum were subsequently assayed for formation (5-100 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase. Protein kinase mediated phosphorylation of skeletal sarcoplasmic reticulum resulted in pronounced stimulation of initial rates and levels of E approximately P in sarcoplasmic reticulum preincubated with either ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) prior to assay (Ca2+-free sarcoplasmic reticulum), or with calcium/EGTA buffer (Ca2+-bound sarcoplasmic reticulum). These effects were evident within a wide range of ionized Ca2+. Phosphorylation of skeletal sarcoplasmic reticulum by protein kinase also increased the initial rate of E approximately P decomposition. These findings suggest that protein kinase-dependent phosphorylation of skeletal sarcoplasmic reticulum regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the active calcium transport observed at steady state.     

Distinct immunopeptide maps of the sarcoplasmic reticulum Ca2+ release channel in malignant hyperthermia

Sarcoplasmic reticulum isolated from malignant hyperthermia-susceptible (MHS) muscle exhibits abnormalities in the regulation of calcium release. To identify the molecular basis of this abnormality, the Ca2+ release channel from both normal and MHS sarcoplasmic reticulum was examined using proteolytic digestion followed by immunoblot staining with a polyclonal antibody against the rabbit Ca2+ release channel protein. Under appropriate conditions, trypsin digestion of isolated sarcoplasmic reticulum vesicles from the two types of pigs revealed a distinct difference in the immunostaining pattern of the Ca2+ release channel-derived peptides. An approximate 86-kDa peptide was the predominant fragment in normal sarcoplasmic reticulum while an approximate 99-kDa peptide fragment was the major peptide detected in MHS sarcoplasmic reticulum. Digestion of sarcoplasmic reticulum vesicles isolated from four normal and four MHS pigs showed that the differences were highly reproducible. Trypsin digestion of sarcoplasmic reticulum isolated from heterozygous pigs, which contain one normal and one MHS allele, showed an antibody staining pattern that was intermediate between MHS and normal sarcoplasmic reticulum. These results can be explained by a primary amino acid sequence difference between the normal and MHS Ca2+ release channels and support the hypothesis that a mutation in the gene coding for the sarcoplasmic reticulum Ca2+ release channel is responsible for malignant hyperthermia.     

Modification of calcium fluxes by dimethyl sulfoxide and 2-butoxyethanol in sarcoplasmic reticulum vesicles: a possible mechanism for skeletal muscle relaxation induced by dimethyl sulfoxide

In order to investigate the mechanism of skeletal muscle relaxation induced by dimethyl sulfoxide, 2-butoxyethanol and dimethyl sulfoxide were examined for their effects on 1) Ca2+ uptake into and efflux from sarcoplasmic reticulum vesicles prepared from rabbit fast skeletal muscle and crayfish tail muscle by the murexide method, 2) ATPase activities of rabbit reticulum vesicles, 3) the isolated phrenic nerve-diaphragm preparation of the rat and 4) crayfish opener muscle preparation. Ca2+ efflux rate from rabbit reticulum vesicles was markedly decreased with increasing concentrations (5-20% v/v) of dimethyl sulfoxide without affecting the maximum Ca2+ uptake by the reticulum. 2-Butoxyethanol showed quite contrary effects. Dimethyl sulfoxide strongly inhibited the activity of basal ATPase rather than of Ca2+-dependent ATPase. 2-Butoxyethanol did not significantly inhibit the activity of basal ATPase, but markedly increased Ca2+-dependent ATPase activity. Antagonisms between dimethyl sulfoxide and caffeine were demonstrated either in contractions of crayfish opener muscles or in the Ca2+ release from crayfish sarcoplasmic reticulum vesicles. These results indicate a possibility that dimethyl sulfoxide reversibly induces skeletal muscle relaxation mainly in the sarcoplasmic reticulum by means of decreasing the rate and the amount of Ca2+ release from the reticulum.     

The role of creatine phosphokinase in supplying energy for the calcium pump system of heart sarcoplasmic reticulum.

An investigation of isolated and purified heart sarcoplasmic reticulum performed in the current study indicates the presence of significant creatine phosphokinase (CPK) activity in this preparation. The localization of CPK on the membrane of sarcoplasmic reticulum has been revealed also by an electron microscopic histochemical method. Under the conditions of the Ca(2+)-ATPase reaction in the presence of creatine phosphate, the release of creatine into the reaction medium is observed, the rate of the latter process being dependent on the MgATP concentration in accordance with the kinetic parameters of the Ca2+-ATPase reaction. CPK localized on the reticular membrane is able to maintain the high rate of calcium consumption by the sarcoplasmic reticulum vesicles. The results obtained demonstrate the close functional coupling between CPK and Ca2+-ATPase in the membrane of sarcoplasmic reticulum and indicate the important functional role of CPK in supplying energy for the Ca(2+)-ATPase reaction and ion transport across the membrane of heart sarcoplasmic reticulum.     

Phospholamban involvement in the maintenance of basal calcium transport in cardiac sarcoplasmic reticulum

Phosphorylation of cardiac sarcoplasmic reticulum membrane vesicles by exogenous c-AMP and c-AMP-dependent protein kinase stimulates calcium uptake and Ca2+-dependent ATP hydrolysis by 40-50% and results in the incorporation of 32P into a 22-KDa protein, phospholamban. Treatment of the membrane with DOC (0.0002% or 5 X 10(-6) M) solubilizes phospholamban from the membrane and induces a 90% inhibition of basal calcium uptake. This inhibition cannot be attributed to an alteration in vesicle integrity or membrane permeability. The (Ca2+ + Mg2+)-ATPase remains associated with the membrane fraction and exhibits optimal levels of Ca2+-stimulated ATP hydrolysis. Phosphorylation prior to DOC treatment allows retention of the phospholamban in the membrane, concomitant with maintenance of the calcium transport activity. The results presented suggest that phospholamban is involved in the maintenance of basal calcium transport function in cardiac sarcoplasmic reticulum and that its phosphorylation stimulates Ca2+ transport.     

Inhibitors of Ca2+ release from the isolated sarcoplasmic reticulum. II. The effects of dantrolene on Ca2+ release induced by caffeine, Ca2+ and depolarization

The effects of dantrolene, which is a known muscle relaxant, on Ca2+ release from the isolated sarcoplasmic reticulum induced by several different methods [1) addition of caffeine, (2) Ca2+ jump, and (3) membrane-depolarization produced by choline chloride replacement of potassium gluconate) were investigated. Dantrolene inhibited caffeine-induced Ca2+ release with C1/2 = 2.5 microM, whereas there was no effect on Ca2+ release induced by a Ca2+ jump. The amount of Ca2+ released by depolarization was reduced if Ca2+ release was triggered in an earlier phase of the steady state of Ca2+ uptake (time elapsed between the addition of ATP and the triggering of Ca2+ release, tATP less than 4 min); while, if triggered in a latter phase (tATP greater than 4 min) dantrolene enhanced depolarization-induced Ca2+ release. C1/2 for the inhibition and that for enhancement of depolarization-induced Ca2+ release were 1.0 and 0.3 microM, respectively. These results suggest that dantrolene affects several different steps of the mechanism by which Ca2+ release is triggered. The sarcoplasmic reticulum and T-tubule membrane fractions had 7.9 nmol dantrolene-binding sites/mg (Kassoc = 1.0 X 10(5) M-1) and 21.0 nmol/mg (Kassoc = 1.1 X 10(5) M-1), respectively. The time-course of dantrolene binding to sarcoplasmic reticulum was monophasic, while that to T-tubules was biphasic.     

Changes in luminal pH caused by calcium release in sarcoplasmic reticulum vesicles.

Fast (milliseconds) Ca2+ release from sarcoplasmic reticulum is an essential step in muscle contraction. To electrically compensate the charge deficit generated by calcium release, concomitant fluxes of other ions are required. In this study we investigated the possible participation of protons as counterions during calcium release. Triad-enriched sarcoplasmic reticulum vesicles, isolated from rabbit fast skeletal muscle, were passively loaded with 1 mM CaCl2 and release was induced at pCa = 5.0 and pH = 7.0 in a stopped-flow fluorimeter. Accompanying changes in vesicular lumen pH were measured with a trapped fluorescent pH indicator (pyranin). Significant acidification (approximately 0.2 pH units) of the lumen occurred within the same time scale (t(1/2) = 0.75 s) as calcium release. Enhancing calcium release with ATP or the ATP analog 5'-adenylylimidodiphosphate (AMPPNP) produced >20-fold faster acidification rates. In contrast, when calcium release induced with calcium with or without AMPPNP was blocked by Mg2+, no acidification of the lumen was observed. In all cases, rate constants of luminal acidification corresponded with reported values of calcium release rate constants. We conclude that proton fluxes account for part (5-10%) of the necessary charge compensation during calcium release. The possible relevance of these findings to the physiology of muscle cells is discussed.     

Is a guanine nucleotide-binding protein involved in excitation-contraction coupling in skeletal muscle?

Plasma membrane depolarization causes skeletal muscle contraction by triggering Ca2+ release from an intracellular membrane network, the sarcoplasmic reticulum. A specialized portion of the sarcoplasmic reticulum, the terminal cisternae, is junctionally associated with sarcolemmal invaginations called the transverse tubules, but the mechanism by which the action potential at the level of the transverse tubules is coupled to Ca2+ release from the terminal cisternae is still mysterious. Here we show that: (i) GTP gamma S, a non-hydrolyzable analog of GTP, elicits isometric force development in skinned muscle fibre; (ii) GTP gamma S is unable to release CA2+ from isolated sarcoplasmic reticulum fractions; (iii) the threshold for tension development is shifted to higher GTP gamma S concentrations by pre-incubation with pertussis toxin. These results suggest that a GTP-binding protein is involved in coupling the action potential of transverse tubules to Ca2+ release from the terminal cisternae.     

Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor

Triadin has been shown to co-localize with the ryanodine receptor in the sarcoplasmic reticulum membrane. We show that immunoprecipitation of solubilized sarcoplasmic reticulum membrane with antibodies directed against triadin or ryanodine receptor, leads to the co-immunoprecipitation of ryanodine receptor and triadin. We then investigated the functional importance of the cytoplasmic domain of triadin (residues 1-47) in the control of Ca2+ release from sarcoplasmic reticulum. We show that antibodies directed against a synthetic peptide encompassing residues 2-17, induce a decrease in the rate of Ca2+ release from sarcoplasmic reticulum vesicles as well as a decrease in the open probability of the ryanodine receptor Ca2+ channel incorporated in lipid bilayers. Using surface plasmon resonance spectroscopy, we defined a discrete domain (residues 18-46) of the cytoplasmic part of triadin interacting with the purified ryanodine receptor. This interaction is optimal at low Ca2+ concentration (up to pCa 5) and inhibited by increasing calcium concentration (IC50 of 300 microM). The direct molecular interaction of this triadin domain with the ryanodine receptor was confirmed by overlay assay and shown to induce the inhibition of the Ca2+ channel activity of purified RyR in bilayer. We propose that this interaction plays a critical role in the control, by triadin, of the Ca2+ channel behavior of the ryanodine receptor and therefore may represent an important step in the regulation process of excitation-contraction coupling in skeletal muscle.     

The effect of calcium ion transport ATPase upon the passive calcium ion permeability of phospholipid vesicles.

The uptake and release of Ca2+ by sarcoplasmic reticulum fragments and reconstituted ATPase vesicles was measured by a stopped-flow fluorescence method using chlortetracycline as Ca2+ indicator. Incorporation of the Ca2+ transport ATPase into phospholipid bilayers of widely different fatty acid composition increases their passive permeability to Ca2+ by several orders of magnitude. Therefore in addition to participating in active Ca2+ transport, the (Mg2+ + Ca2+)-activated ATPase also forms hydrophilic channels across the membrane. The relative insensitivity of the permeability effect of ATPase to changes in the fatty acid composition of the membrane is in accord with the suggestion that the Ca2+ channels arise by protein-protein interaction between four ATPase molecules. The reversible formation of these channels may have physiological significance in the rapid Ca2+ release from the sarcoplasmic reticulum during activation of muscle.     

Effect of pH on calcium accumulation and release by isolated fragments of cardiac and skeletal muscle sarcoplasmic reticulum.

The ability of a sudden increase in pH to initiate a release of calcium from isolated skeletal and cardiac muscle sarcoplasmic reticulum following calcium accumulation in the absence of a precipitating anion (calcium binding) is described. In skeletal sarcoplasmic reticulum a sudden increase in pH caused a rapid release of accumulated calcium. In cardiac sarcoplasmic reticulum a sudden increase in pH before the calcium binding process was complete caused the release of a small amount of calcium at a relatively slow rate. A sudden change in pH after the completion of calcium binding failed to trigger a release of calcium. The effect of pH on oxalate supported calcium uptake and on unidirectional calcium efflux rate by cardiac sarcoplasmic reticulum was also studied. Both the rate of calcium uptake and of unidirectional calcium efflux increased as the pH was raised from 6.4 to 7.2, reflecting an increased permeability of the sarcoplasmic reticulum membrane to calcium. These results indicate that in cardiac muscle a sudden increase in pH is unlikely to be the in vivo signal for calcium release from the sarcoplasmic reticulum. However, the effect of pH on calcium uptake and efflux by cardiac sarcoplasmic reticulum may contribute to the negative inotropic effect of an acidosis on the heart.     

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds

The caffeine-sensitive Ca2+ release pathway in skeletal muscle was identified and characterized by studying the release of 45Ca2+ from heavy sarcoplasmic reticulum (SR) vesicles and by incorporating the vesicles or the purified Ca2+ release channel protein complex into planar lipid bilayers. First-order rate constants for 45Ca2+ efflux of 1 s-1 were obtained in the presence of 1-10 microM free Ca2+ or 2 X 10(-9) M free Ca2+ plus 20 mM caffeine. Caffeine- and Ca2+-induced 45Ca2+ release were potentiated by ATP and Mg.ATP, and were both inhibited by Mg2+. Dimethylxanthines were similarly (3,9-dimethylxanthine) or more (1,7-, 1,3-, and 3,7-dimethylxanthine) effective than caffeine in increasing the 45Ca2+ efflux rate. 1,9-Dimethylxanthine and 1,3-dimethyluracil (which lacks the imidazole ring) did not appreciably stimulate 45Ca2+ efflux. Recordings of calcium ion currents through single channels showed that the Ca2+- and ATP-gated SR Ca2+ release channel is activated by addition of caffeine to the cis (cytoplasmic) and not the trans (lumenal) side of the channel in the bilayer. The single channel measurements further revealed that caffeine activated Ca2+ release by increasing the number and duration of open channel events without a change of unit conductance (107 pS in 50 mM Ca2+ trans). These results suggest that caffeine exerts its Ca2+ releasing effects in muscle by activating the high-conductance, ligand-gated Ca2+ release channel of sarcoplasmic reticulum.     

The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes

Ryanodine at concentrations of 0.01-10 microM increased, while greater concentrations of 10-300 microM decreased the calcium permeability of both rabbit fast twitch skeletal muscle junctional and canine cardiac sarcoplasmic reticulum membranes. Ryanodine did not alter calcium binding by either sarcoplasmic reticulum membranes or the calcium binding protein, calsequestrin. Therefore, the effects by this agent appear to involve only changes in membrane permeability, and the characteristics of the calcium permeability pathway affected by ryanodine were those of the calcium release channel. Consistent with this, the actions by ryanodine were localized to junctional sarcoplasmic reticulum membranes and were not observed with either longitudinal sarcoplasmic reticulum or transverse tubular membranes. In addition, passage of the junctional sarcoplasmic reticulum membranes through a French press did not diminish the effects of ryanodine indicating that intact triads were not required. Under the conditions used for the permeability studies, the binding of [3H]ryanodine to skeletal junctional sarcoplasmic reticulum membranes was specific and saturable, and Scatchard analyses indicated the presence of a single binding site with a Kd of 150-200 nM and a maximum capacity of 10.1-18.9 pmol/mg protein. [3H]ryanodine binding to this site and the increase in membrane calcium permeability caused by low concentrations of ryanodine had similar characteristics suggesting that actions at this site produce this effect. Depending on the assay conditions used, ryanodine (100-300 microM) could either increase or decrease ATP-dependent calcium accumulation by skeletal muscle junctional sarcoplasmic reticulum membranes indicating that the alterations of sarcoplasmic reticulum membrane calcium permeability caused by this agent can be determined in part by the experimental environment.