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.     

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.     

Calcium release from sarcoplasmic reticulum of normal and dystrophic mice

Contraction of skeletal muscle is triggered by release of calcium from the sarcoplasmic reticulum. In this study, highly purified normal and dystrophic mouse sarcoplasmic reticulum vesicles were compared with respect to calcium release characteristics. Sarcoplasmic reticulum vesicles were actively loaded with calcium in the presence of an ATP-regenerating system. Calcium fluxes were followed by dual wavelength spectrophotometry using the metallochromic indicators antipyrylazo III and arsenazo III, and by isotopic techniques. Calcium release from sarcoplasmic reticulum vesicles was elicited by (a) changing the free calcium concentration of the assay medium (calcium-induced calcium release); (b) addition of a permeant anion to the assay medium, following calcium loading in the presence of a relatively impermeant anion (depolarization-induced calcium release); (c) addition of the lipophilic anion tetraphenylboron (TPB^?) to the assay medium and (d) using specific experimental conditions, i.e. high phosphate levels and low magnesium (spontaneous calcium release). Drugs known to influence Ca²⁺ release were shown to differentially affect the various types of calcium release. Caffeine (10 mM) was found to enhance calcium-induced calcium release from isolated sarcoplasmic reticulum. Ruthenium red (20 μM) inhibited both calcium-induced calcium release and tetraphenylboron-induced calcium release, and partially inhibited spontaneous calcium release and depolarization-induced calcium release. Local anesthetics inhibited spontaneous calcium release in a time-dependent manner, and inhibited calcium-induced calcium release instantaneously, but did not inhibit depolarization-induced calcium release. Use of pharmacological agents indicates that several types of calcium release operate in vitro. No significant differences were found between normal and dystrophic sarcoplasmic reticulum in calcium release kinetics or drug sensitivities.     

Lack of effects of calcium X calmodulin-dependent phosphorylation on Ca2+ release from cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium X calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca2+ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium X calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca2+ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium X calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca2+ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent 45Ca2+-40Ca2+ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium X calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca2+ release and 45Ca2+-40Ca2+ exchange.     

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.     

Calcium transport in transverse tubules isolated from rabbit skeletal muscle

Isolated transverse tubule vesicles free of sarcoplasmic reticulum transport calcium with high affinity in the presence of ATP. The calcium transport by transverse tubules differs from calcium transport by sarcoplasmic reticulum. It is not increased by oxalate or phosphate, it has a different temperature dependence, it is inhibited by sub-micromolar concentrations of orthovanadate, it is stimulated by calmodulin, and is inhibited by quercetin without causing calcium release. The rates of calcium transport by transverse tubules are two orders of magnitude lower than those of sarcoplasmic reticulum, suggesting that the calcium pump protein of transverse tubules is a minor component of the membrane. Addition of calmodulin to transverse tubule vesicles--treated with high salt in the presence of EGTA to remove endogenous calmodulin--caused a marked stimulation of transport rates at low concentrations of calcium, and decreased from 1.0 to 0.3 microM the calcium concentration at which half-maximal rates of transport were obtained. A role for the transverse tubule calcium pump in maintaining low sarcoplasmic calcium concentrations is proposed.     

Luminal calcium regulation of calcium release from sarcoplasmic reticulum

This article discusses how changes in luminal calcium concentration affect calcium release rates from triad-enriched sarcoplasmic reticulum vesicles, as well as single channel opening probability of the ryanodine receptor/calcium release channels incorporated in bilayers. The possible participation of calsequestrin, or of other luminal proteins of sarcoplasmic reticulum in this regulation is addressed. A comparison with the regulation by luminal calcium of calcium release mediated by the inositol 1,4,5-trisphosphate receptor/calcium channel is presented as well.     

Real-time measurement of the rate of sarcoplasmic reticulum calcium pumping in skinned muscle fibers

A method and a device had been developed to directly measure the accumulation of calcium in the sarcoplasmic reticulum and its release from the sarcoplasmic reticulum, depending on the free Ca²⁺ concentration in the solution. The sarcoplasmic reticulum occupies to 30% of the volume of the swim bladder muscles of the oyster toadfish Opsanus tau. To isolate and skin muscle fibers and to remove the accumulated calcium from the sarcoplasmic reticulum, a set of solutions containing EGTA as a pCa buffer was used. To measure the calcium exchange between a fiber ～10 nl in volume and the solution in a 5-μl cuvette, instead of EGTA, 50–100 μM FURA2 or bisFURA2 was used both as pCa buffer and as a fluorescent indicator of the calcium concentration in the cuvette. An increase in fluorescence intensity meant an increase in the free FURA concentration in the solution surrounding the fiber since the calcium entering the sarcoplasmic reticulum was taken from this solution. The slope of the fluorescence curve corresponded to a rate of calcium accumulation in the sarcoplasmic reticulum of 1.6 μmol per second per liter of the solution in the cuvette or 2.6 mmol per second per liter of the sarcoplasmic reticulum. A solution without oxalate and ruthenium red may exhibit oscillations of the free FURA concentration, which can be explained by calcium-activated calcium release from the sarcoplasmic reticulum.     

Effect of ammonium ions on calcium release from fragmented sarcoplasmic reticulum

It is established that in AMP deamination by sarcoplasmic reticulum fragments there occurs an intensified release of the previously accumulated calcium. UMP has no noticeable effect on this process. The level of accumulated 45Ca+ in the fragmented sarcoplasmic reticulum is decreased when ammonium ions load is introduced into the medium. If the sarcoplasmic reticulum fragments were loaded with 45Ca2+ and then washed off and incubated in the isotonic sucrose solution, the calcium release is more intensified when ammonium ions are introduced into the medium. The results of ultrasound and A23187 treatment of the membranes evidence for the calcium release from the inner space of vesicles.     

Patterns of sarcomere activation, temperature dependence, and effect of ryanodine in chemically skinned cardiac fibers

Functionally skinned and electrochemically shunted myocytes were prepared by perfusing rat hearts with collagenase in order to obtain a technically improved measurement of sarcomere dynamics and to evaluate the role of sarcoplasmic reticulum in situ with respect to contractile activation. In the presence of micromolar calcium, the myocytes exhibited phasic and propagated contraction waves beginning at one end and proceeding along the myocyte. Beating rates, the propagation velocity of the activation wave, and single sarcomere shortening and relaxation velocities were obtained by manual or automated analysis of 16-mm film recorded at 170 frames/s from a camera attached to a microscope that was equipped with a temperature-controlled stage. In parallel experiments, calcium accumulation by the sarcoplasmic reticulum of the myocytes in situ was measured by direct isotopic tracer methods. The frequency (10-38 min-1) of spontaneous contractions, the velocity (1.9-7.4 microns . s-1) of sarcomere shortening, and the velocity (1.7-6.8 microns . s-1) of sarcomere relaxation displayed identical temperature dependences (Q10 = 2.2), which are similar to that of the calcium pump of sarcoplasmic reticulum and are consistent with a rate limit imposed by enzyme-catalyzed mechanisms on all these parameters. On the other hand, the velocity (77- 159 microns . s-1) of sequential sarcomere activation displayed a lower temperature dependence (Q10 = 1.5), which is consistent with a diffusion-limited and self-propagating release of calcium from one sarcomere to the other. The phasic contractile activity of the dissociated myocytes was inhibited by 10(-8)-10(6) M ryanodine (and not by myolemmal calcium blockers) under conditions in which calcium accumulation by sarcoplasmic reticulum in situ was demonstrated to proceed optimally. The effect of ryanodine is attributed to an interaction of this drug with sarcotubular structures, producing inhibition of calcium release from the sarcoplasmic reticulum. The consequent lack of sarcomere activation underlines the role of sarcoplasmic reticulum uptake and release in the phasic contractile activation of the electrochemically shunted myocytes.     

Passive Ca2+ permeability of phospholipid vesicles and sarcoplasmic reticulum membranes.

During the excitation of muscle the estimated rate of Ca2+ release from sarcoplasmic reticulum may increase 10(3)- to 10(4)-fold compared with relaxed muscle or isolated sarcoplasmic reticulum in vitro, implying a major change in the calcium permeability of the sarcoplasmic reticulum membrane. As a first step in the assessment of the role of various membrane constituents in the regulation of calcium fluxes, the contribution of phospholipids to the definition of calcium permeability was studied in model systems. The rate of calcium release from vesicles prepared from pure phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositides, cardiolipin, and extracted microsomal lipids is in the range of 10(-15) to 10(18) mol of calcium/cm2/s. This rate is several orders of magnitude lower than the passive calcium outflux from isolated sarcoplasmic reticulum membranes. The permeability to Ca2+ is influenced by fatty acid composition and net charge and it is markedly increased with increasing temperature or after the addition of local anesthetics.     

Reconstitution of an active calcium pump in sarcoplasmic reticulum.

Recovery of calcium transport and calcium-activated ATPase activity was studied in relation to the retention of protein components in sarcoplasmic reticulum reconstituted after solubilization with deoxycholate and centrifugation, followed by removal of the detergent from the supernatant by dialysis. Control sarcoplasmic reticulum was similarly treated except for omission of deoxycholate. Maximum capacity for oxalate- and phosphate-supported calcium uptake was increased 2- to 3-fold in reconstituted sarcoplasmic reticulum compared to original and control. Calcium uptake velocity of the reconstituted sarcoplasmic reticulum was approximately 80% that of original and 90% of control sarcoplasmic reticulum. Calcium uptake/ATP hydrolysis ratio was approximately 2 in the original sarcoplasmic reticulum and decreased to approximately 1 in the control and reconstituted sarcoplasmic reticulum. Calcium storage in the absence of calcium-precipitating anion was approximately 85% in control and 70% in reconstituted sarcoplasmic reticulum, compared to the original sarcoplasmic reticulum. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid-induced calcium release after phosphate-supported calcium uptake was slower in reconstituted sarcoplasmic reticulum than in original or control sarcoplasmic reticulum. Polyacrylamide gel electrophoresis of original and control sarcoplasmic reticulum showed similar amounts of protein components of approximately 93,000, 59,000, 50,000, 30,000 to 37,000, and 20,000 to 26,000 daltons. Reconstituted sarcoplasmic reticulum, however, lost over 85% of the 50,000- and 20,000- to 26,000-dalton proteins while retaining most of its calcium transport functions.     

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.     

Activation of skinned muscle fibers by calcium and strontium ions

Intact and mechanically skinned skeletal muscle fibers of the crab Carcinus maenas have been used. The aim of the experiments was to determine the origin of the mechanical activity recorded in intact crab muscle fibers exhibiting an inward strontium current in strontium solution without calcium. To do so, the effect of strontium ions in inducing activation of contractile proteins and calcium release from the sarcoplasmic reticulum has been studied. The properties of the sarcoplasmic reticulum membrane towards strontium ions, i.e., the efficiency of the calcium ATPase towards strontium ions and the capability to release strontium ions have been investigated. Results show that the contractile proteins have a lower affinity for strontium than for calcium ions. However, the maximum bound strontium is identical to the maximum bound calcium. As for the sarcoplasmic reticulum, strontium ions can induce a calcium release and also can be taken up by the calcium ATPase and be released. We concluded that the mechanical activity in intact fibers bathed in a strontium medium has two origins: first, a direct and partial activation of the contractile proteins by strontium ions flowing through the calcium channel; second, a contractile proteins activation of calcium ions released by the sarcoplasmic reticulum by a "strontium-induced calcium release" mechanism.     

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.     

Lack of effects of calcium · calmodulin-dependent phosphorylation on Ca2+ release from cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium · calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca²⁺ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium · calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca²⁺ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium · calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca²⁺ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent ⁴⁵Ca²⁺ − ⁴⁰Ca²⁺ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium · calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca²⁺ release and ⁴⁵Ca²⁺ − ⁴⁰Ca²⁺ exchange.     

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic sarcoplasmic reticulum vesicles

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic smooth muscle sarcoplasmic reticulum vesicles was examined using the calcium indicator antipyrylazo III. Calcium release was initiated by addition of inositol 1,4,5-trisphosphate (IP3) to aortic vesicles 7 min after initiation of ATP-supported calcium uptake. Half-maximal calcium release occurred at 1 microM IP3, with maximal calcium release amounting to 25 +/- 2% of the intravesicular calcium (n = 12, 9 preparations). Ruthenium red (10-20 microM), which has been reported to block IP3-induced calcium release from skeletal muscle sarcoplasmic reticulum, did not inhibit aortic IP3-induced calcium release. Elevation of Mg2+ concentration from 0.06 to 7.8 mM inhibited aortic IP3-induced calcium release 75%, which contrasts with the Mg2+-insensitive IP3-induced calcium release from platelet reticular membranes. The IP3-dependence of aortic calcium release suggested that Mg2+ acted as a noncompetitive inhibitor. Thus, aortic sarcoplasmic reticulum vesicles contain an IP3-sensitive calcium pathway which is inhibited by millimolar concentrations of Mg2+, but which is not inhibited by Ruthenium red and so differs from the previously described IP3-sensitive calcium pathways in skeletal muscle and platelet reticular membranes.     

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.     

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.     

Palmitylcarnitine inhibition of the calcium pump in cardiac sarcoplasmic reticulum: a possible role in myocardial ischemia.

Palmitylcarnitine is a time-dependent inhibitor of the Ca²⁺-ATPase activity of cardiac sarcoplasmic reticulum isolated from adult dogs. Half-maximal inhibition was obtained at approximately 20 μM (2 μmoles/mg). The extent of inhibition depended on the ratio of palmitylcarnitine to sarcoplasmic reticulum protein. Calcium uptake by cardiac sarcoplasmic reticulum (measured in the presence of sodium oxalate) was found to be even more sensitive to inhibition by palmitylcarnitine and complete inhibition was obtained at concentrations as low as 2.5 μM (0.25 μmole/mg) following preincubation. Calcium binding (measured in the absence of oxalate) was inhibited by palmitylcarnitine and calcium release was stimulated at similar ratios. The level of palmitylcarnitine has been reported to increase several fold in myocardial ischemia and inhibition of the sarcoplasmic reticulum calcium pump could conceivably contribute either to the initial loss of contractility or the subsequent inability to restore full contractile function after prolonged ischemia.