Characterization of Ca2+ release from heterogeneous Ca2+ stores in sarcoplasmic reticulum isolated from arterial and gastric smooth muscle

Ca2+ transport was investigated in vesicles of sarcoplasmic reticulum subfractionated from bovine main pulmonary artery and porcine gastric antrum using digitonin binding and zonal density gradient centrifugation. Gradient fractions recovered at 15-33% sucrose were studied as the sarcoplasmic reticulum component using Fluo-3 fluorescence or 45Ca2+ Millipore filtration. Thapsigargin blocked active Ca2+ uptake and induced a slow Ca2+ release from actively loaded vesicles. Unidirectional 45Ca2+ efflux from passively loaded vesicles showed multicompartmental kinetics. The time course of an initial fast component could not be quantitatively measured with the sampling method. The slow release had a half-time of several minutes. Both components were inhibited by 20 microM ruthenium red and 10 mM Mg2+. Caffeine, inositol 1,4,5-trisphosphate, ATP, and diltiazem accelerated the slow component. A Ca2+ release component activated by ryanodine or cyclic adenosine diphosphate ribose was resolved with Fluo-3. Comparison of tissue responses showed that the fast Ca2+ release was significantly smaller and more sensitive to inhibition by Mg2+ and ruthenium red in arterial vesicles. They released more Ca2+ in response to inositol 1,4,5-trisphosphate and were more sensitive to activation by cyclic adenosine diphosphate ribose. Ryanodine and caffeine, in contrast, were more effective in gastric antrum. In each tissue, the fraction of the Ca2+ store released by sequential application of caffeine and inositol 1,4,5-trisphosphate depended on the order applied and was additive. The results indicate that sarcoplasmic reticulum purified from arterial and gastric smooth muscle represents vesicle subpopulations that retain functional Ca2+ channels that reflect tissue-specific pharmacological modulation. The relationship of these differences to physiological responses has not been determined.     

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca²⁺ transients and the rate of Ca²⁺ release (dCa_REL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca²⁺ indicators. Single pulse experiments with different returning potentials showed that Ca²⁺ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca²⁺ removal (dCa_REM/dt) was studied by fitting the decaying phase of the Ca²⁺ transient (Melzer, Ríos & Schneider, 1986) and dCa_REL/dt was calculated as the difference between dCa/dt and dCa_REM/dt. The fast Ca²⁺ release decayed as a consequence of Ca²⁺ inactivation of Ca²⁺ release. Double pulse experiments showed inactivation of the fast Ca²⁺ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca²⁺ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca²⁺]_i and the inhibited amount of Ca²⁺ release was 0.98. The [Ca²⁺]_i for 50% inactivation of dCa_REL/dt was 0.25 m, and the minimum number of sites occupied by Ca²⁺ to inactivate the Ca²⁺ release channel was 3.0. These data support Ca²⁺ binding and inactivation of SR Ca²⁺ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.     

Ca2+-induced sarcoplasmic reticulum Ca2+ release in myotubularin-deficient muscle fibers

Skeletal muscle deficiency in the 3-phosphoinositide (PtdInsP) phosphatase myotubularin (MTM1) causes myotubular myopathy which is associated with severe depression of voltage-activated sarcoplasmic reticulum Ca²⁺ release through ryanodine receptors. In the present study we aimed at further understanding how Ca²⁺ release is altered in MTM1-deficient muscle fibers, at rest and during activation. While in wild-type muscle fibers, SR Ca²⁺ release exhibits fast stereotyped kinetics of activation and decay throughout the voltage range of activation, Ca²⁺ release in MTM1-deficient muscle fibers exhibits slow and unconventional kinetics at intermediate voltages, suggestive of partial loss of the normal control of ryanodine receptor Ca²⁺ channel activity. In addition, the diseased muscle fibers at rest exhibit spontaneous elementary Ca²⁺ release events at a frequency 30 times greater than that of control fibers. Eighty percent of the events have spatiotemporal properties of archetypal Ca²⁺ sparks while the rest take either the form of lower amplitude, longer duration Ca²⁺ release events or of a combination thereof. The events occur at preferred locations in the fibers, indicating spatially uneven distribution of the parameters determining spontaneous ryanodine receptor 1 opening. Spatially large Ca²⁺ release sources were obviously involved in some of these events, suggesting that opening of ryanodine receptors in one cluster can activate opening of ryanodine receptors in a neighboring one. Overall results demonstrate that opening of Ca²⁺-activated ryanodine receptors is promoted both at rest and during excitation-contraction coupling in MTM1-deficient muscle fibers. Because access to this activation mode is denied to ryanodine receptors in healthy skeletal muscle, this may play an important role in the associated disease situation.     

The sarcoplasmic reticulum Ca2+ store arrangement in vascular smooth muscle

In vascular smooth muscle cells, Ca²⁺ release via IP₃ receptors (IP₃R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) Ca²⁺ store contributes significantly to the regulation of cellular events such as gene regulation, growth and contraction. Ca²⁺ release from various regions of a structurally compartmentalized SR, it is proposed, may selectively activate different cellular functions. Multiple SR compartments with various receptor arrangements are proposed also to exist at different stages of smooth muscle development and in proliferative vascular diseases such as atherosclerosis. The conclusions on SR organization have been derived largely from the outcome of functional studies. This study addresses whether the SR Ca²⁺ store is a single continuous interconnected network or multiple separate Ca²⁺ pools in single vascular myocytes. To do this, the consequences of depletion of the SR in small restricted regions on the Ca²⁺ available throughout the store was examined using localized photolysis of caged-IP₃ and focal application of ryanodine in guinea-pig voltage-clamped single portal vein myocytes. From one small site on the cell, the entire SR could be depleted via either RyR or IP₃R. The entire SR could also be refilled from one small site on the cell. The results suggest a single luminally continuous SR exists. However, the opening of IP₃R and RyR was regulated by the Ca²⁺ concentration within the SR (luminal [Ca²⁺]). As the luminal [Ca²⁺] declines, the opening of the receptors decline and stop, and there may appear to be stores with either only RyR or only IP₃R. The SR Ca²⁺ store is a single luminally continuous entity which contains both IP₃R and RyR and within which Ca²⁺ is accessed freely by each receptor. While the SR is a single continuous entity, regulation of IP₃R and RyR by luminal [Ca²⁺] explains the appearance of multiple stores in some functional studies.     

GTP requirement for inositol-1,4,5-trisphosphate-induced Ca2+ release from sarcoplasmic reticulum in smooth muscle

We have examined inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release from the sarcoplasmic reticulum (SR) in the skinned vascular smooth muscle. The amount of Ca2+ in the SR was estimated indirectly by caffeine-induced contraction of the skinned preparation. The Ca2+ release from the SR by IP3 required GTP. A non-hydrolyzable analogue of GTP, guanosine 5'-(beta gamma-imido) triphosphate (GppNHp) could substitute for GTP in the IP3-induced Ca2+ release. These results suggest an involvement of GTP-binding protein in the mechanism of Ca2+ release from the SR by IP3 in smooth muscle.     

Regulation of sarcoplasmic reticulum Ca2+ reuptake in porcine airway smooth muscle

Regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in airway smooth muscle (ASM) during agonist stimulation involves sarcoplasmic reticulum (SR) Ca(2+) release and reuptake. The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is key to replenishment of SR Ca(2+) stores. We examined regulation of SERCA in porcine ASM: our hypothesis was that the regulatory protein phospholamban (PLN) and the calmodulin (CaM)-CaM kinase (CaMKII) pathway (both of which are known to regulate SERCA in cardiac muscle) play a role. In porcine ASM microsomes, we examined the expression and extent of PLN phosphorylation after pharmacological inhibition of CaM (with W-7) vs. CaMKII (with KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In parallel experiments using enzymatically dissociated single ASM cells loaded with the Ca(2+) indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca(2+)](i) responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca(2+)](i) transients to 1 microM ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W-7 or KN-62 did not prevent initiation of ACh-induced [Ca(2+)](i) oscillations (which were previously shown to result from repetitive SR Ca(2+) release/reuptake). However, when ACh-induced [Ca(2+)](i) oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca(2+)](i) transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca(2+)](i) oscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR Ca(2+) release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca(2+) reuptake, potentially through altered PLN phosphorylation.     

Acidosis facilitates spontaneous sarcoplasmic reticulum Ca2+ release in rat myocardium

Previous studies have shown that acidosis increases myoplasmic [Ca2+] (Cai). We have investigated whether this facilitates spontaneous sarcoplasmic reticulum (SR) Ca2+ release and its functional sequelae. In unstimulated rat papillary muscles, exposure to an acid solution (produced by increasing the [CO2] of the perfusate from 5 to 20%) caused a rapid increase in the mean tissue Cai, as measured by the photoprotein aequorin. This was paralleled by an increase in spontaneous microscopic tissue motion caused by localized Ca2+ myofilament interactions, as monitored in fluctuations in the intensity of laser light scattered by the muscle. In regularly stimulated muscles, acidosis increased the size of the Ca2+ transient associated with each contraction and caused the appearance of Cai oscillations in the diastolic period. In unstimulated single myocytes, acidosis depolarized the resting membrane potential by approximately 5 mV and enhanced the frequency of spontaneous contractile waves. The small sarcolemmal depolarization associated with each contractile wave increased and occasionally initiated spontaneous action potentials. In regularly stimulated myocytes, acidosis caused de novo spontaneous contractile waves between twitches; these waves were associated with a decrease in the amplitude of the subsequent stimulated twitch. Ryanodine (2 microM) abolished all evidence of spontaneous Ca2+ release during acidosis, markedly reduced the acidosis-induced increase in aequorin light, and reduced resting tension. We conclude that acidosis increases the likelihood for the occurrence of spontaneous SR Ca2+ release, which can cause spontaneous action potentials, increase resting tension, and negatively affect twitch tension.     

Effect of halothane on Ca2+-induced Ca2+ release from sarcoplasmic reticulum vesicles isolated from rat skeletal muscle

Halothane induces the release of Ca2+ from a subpopulation of sarcoplasmic reticulum vesicles that are derived from the terminal cisternae of rat skeletal muscle. Halothane-induced Ca2+ release appears to be an enhancement of Ca2+-induced Ca2+ release. The low-density sarcoplasmic reticulum vesicles which are believed to be derived from nonjunctional sarcoplasmic reticulum lack the capability of both Ca2+-induced and halothane-induced Ca2+ release. Ca2+ release from terminal cisternae vesicles induced by halothane is inhibited by Ruthenium red and Mg2+, and require ATP (or an ATP analogue), KCl (or similar salt) and extravesicular Ca2+. Ca2+-induced Ca2+ release has similar characteristics.     

Coordination between Ca2+ release and subsequent re-uptake in the sarcoplasmic reticulum

We here report the results of our recent effort to produce, in the isolated sarcoplasmic reticulum (SR), a biphasic Ca2+ release and Ca2+ re-uptake transient and to resolve the kinetic relationship between Ca2+ release and re-uptake of the released Ca2+. Ca2+ release from the SR was induced by polylysine (the ryanodine receptor-specific Ca2+ release trigger) at various levels of calcium loading, or at various doses of the trigger. The changes in the Ca2+ concentration in the reaction solution and in the lumenal Ca2+ concentration were determined by stopped-flow spectroscopy using fluo-3 and mag-fura-2AM, respectively. At higher levels of calcium loading (>150 nmol/mg), polylysine induced monophasic Ca2+ release curves (without an appreciable re-uptake phase) as reported in most studies in the literature. However, lowering the calcium loading level to an intermediate range (100-150 nmol/mg) produced the desired biphasic transient curves consisting of Ca2+ release and Ca2+ re-uptake phases. Under these conditions, the increase in the polylysine concentration resulted in the increase of both the rate of Ca2+ release and that of re-uptake of the released Ca2+. The maximal rate of Ca2+ release and that of re-uptake showed a parallel relationship in the polylysine concentration range of 0-10 microM. This indicates that Ca2+ release from the SR and re-uptake of the released Ca2+ via the SR Ca2+ pump are well-coordinated processes. The changes in the lumenal Ca2+ concentration during the release and re-uptake reaction were monitored at an optimum level of calcium loading while clamping the extravesicular Ca2+ concentration at a constant value. There was again a tight correlation between Ca2+ release (decrease of the lumenal Ca2+ concentration) and re-uptake (increase of the lumenal Ca2+ concentration), indicating that acceleration of the re-uptake is controlled by the rate of decrease of the lumenal Ca2+ concentration. We propose that one of the mechanisms, by which the mode of coordination between the two components of the biphasic Ca2+ transient (viz. Ca2+ release via the ryanodine receptor and Ca2+ re-uptake via the SR Ca2+ pump) is controlled, is the change in the Ca2+ concentration gradient across the SR membrane.     

Intraluminal Ca2+ dependence of Ca2+ and ryanodine-mediated regulation of skeletal muscle sarcoplasmic reticulum Ca2+ release.

The action of ryanodine upon sarcoplasmic reticulum (SR) Ca2+ handling is controversial with evidence for both activation and inhibition of SR Ca2+ release. In this study, the role of the intraluminal SR Ca2+ load was probed as a potential regulator of ryanodine-mediated effects upon SR Ca2+ release. Through dual-wavelength spectroscopy of Ca2+:antipyrylazo III difference absorbance, the intraluminal Ca2+ dependence of ryanodine and Ca(2+)-induced Ca2+ release (CICR) from skeletal SR vesicles was examined. Ryanodine addition after initiation of Ca2+ uptake (a) increased the intraluminal Ca2+ sensitivity of CICR and (b) stimulated spontaneous Ca2+ release with a delayed onset. These ryanodine effects were inversely proportional to the intraluminal Ca2+ load. Ryanodine also inhibited subsequent CICR after reaccumulation of Ca2+ released from the initial CICR. These results provide evidence that ryanodine inhibits transitions between low and high affinity Ca2+ binding states of an intraluminal Ca2+ compartment, possibly calsequestrin. Conformational transitions of calsequestrin may be reciprocally coupled to transitions between open and closed states of the Ca2+ release channel.     

Regulation of cardiac muscle Ca2+ release channel by sarcoplasmic reticulum lumenal Ca2+.

The cardiac muscle sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor) is a ligand-gated channel that is activated by micromolar cytoplasmic Ca2+ concentrations and inactivated by millimolar cytoplasmic Ca2+ concentrations. The effects of sarcoplasmic reticulum lumenal Ca2+ on the purified release channel were examined in single channel measurements using the planar lipid bilayer method. In the presence of caffeine and nanomolar cytosolic Ca2+ concentrations, lumenal-to-cytosolic Ca2+ fluxes >/=0.25 pA activated the channel. At the maximally activating cytosolic Ca2+ concentration of 4 microM, lumenal Ca2+ fluxes of 8 pA and greater caused a decline in channel activity. Lumenal Ca2+ fluxes primarily increased channel activity by increasing the duration of mean open times. Addition of the fast Ca2+-complexing buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cytosolic side of the bilayer increased lumenal Ca2+-activated channel activities, suggesting that it lowered Ca2+ concentrations at cytosolic Ca2+-inactivating sites. Regulation of channel activities by lumenal Ca2+ could be also observed in the absence of caffeine and in the presence of 5 mM MgATP. These results suggest that lumenal Ca2+ can regulate cardiac Ca2+ release channel activity by passing through the open channel and binding to the channel's cytosolic Ca2+ activation and inactivation sites.     

Heavy metal-induced Ca2+ release from sarcoplasmic reticulum

Two distinct forms of Ca2+ release from isolated sarcoplasmic reticulum vesicles in response to additions of heavy metals (silver and mercurials) are described. One form of heavy metal-induced Ca2+ release involves the ruthenium red-sensitive Ca2+ release channel localized in terminal cisternae. The other form of heavy metal-induced Ca2+ release appears to involve all portions of the sarcoplasmic reticulum and is insensitive to ruthenium red. This latter form of Ca2+ release occurs over a similar range of heavy metal concentrations as inhibition of the sarcoplasmic reticulum Ca2+ pump but does not appear to be a result solely of such pump inhibition. Both forms of Ca2+ release are inhibited by glutathione, an endogenous constituent of muscle fibers, and by dithiothreitol, agents which prevent sulfhydryl oxidation. To assess the role of any sulfhydryl oxidation in sarcoplasmic reticulum Ca2+ release physiologically, dithiothreitol and glutathione were introduced inside muscle fibers and effects on excitation-contraction coupling examined. The results strongly suggest that sulfhydryl oxidation plays no essential role in skeletal muscle excitation-contraction coupling.     

Spectroscopic determination of sarcoplasmic reticulum Ca2+ uptake and Ca2+ release

In this report we describe the application of spectroscopic methods to the study of Ca2+ release by isolated native sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle. To date, dual-wavelength spectroscopy of arsenazo III and antipyrylazo III difference absorbance have been the most common spectroscopic methods for the assay of SR Ca2+ transport. The utility of these methods is the ability to manipulate intraluminal Ca2+ loading of SR vesicles. These methods have also been useful for studying the effect of both agonists and antagonists upon SR Ca2+ release and Ca2+ uptake. In this study, we have developed the application of Calcium Green-2, a long-wavelength excitable fluorescent indicator, for the study of SR Ca2+ uptake and release. With this method we demonstrate how ryanodine receptor Ca2+ channel opening and closing is regulated in a complex manner by the relative distribution of Ca2+ between extraluminal and intraluminal Ca2+ compartments. Intraluminal Ca2+ is shown to be a key regulator of Ca2+ channel opening. However, these methods also reveal that the intraluminal Ca2+ threshold for Ca2+-induced Ca2+ release varies as a function of extraluminal Ca2+ concentration. The ability to study how the relative distribution of a finite pool of Ca2+ across the SR membrane influences Ca2+ uptake and Ca2+ release may be useful for understanding how the ryanodine receptor is regulated, in vivo.     

Activation of Ca2+ release in isolated sarcoplasmic reticulum

Summary The relationship between Ca²⁺ release from sarcoplasmic reticulum, induced by elevated pH, tetraphenylboron (TPB^–) or chemical modification, and the change in the surface charge of the membranes as measured by the fluorescence intensity of anilinonaphthalene sulfonate (ANS) is examined. The stimulated Ca²⁺ release is inhibited by dicyclohexylcarbodiimide and external Ca²⁺. TPB^–, but not tetraphenylarsonium (TPA⁺), causes a decrease in ANS^– fluorescence, with 50% decrease occurring at about 5 m TPB^–. The decrease in ANS^– fluorescence as well as the inhibition of Ca²⁺ accumulation induced by TPB^– are prevented by TPA⁺. A linear relationship between the decrease in membrane surface potential and the extent of the Ca²⁺ released by TPB^– is obtained. Similar levels of [³H]TPB^– bound to sarcoplasmic reticulum membranes were obtained regardless of whether or not the vesicles have taken up Ca²⁺. The inhibition of Ca²⁺ accumulation and the [³H]TPB^– incorporation into the membranes were correlated. Ca²⁺ release from sarcoplasmic reticulum, by pH elevation, chemical modification or by addition of NaSCN (0.2 to 0.5m) or the Ca²⁺ ionophore ionomycin, is also accompanied by a decrease in ANS^– fluorescence intensity. However, chemical modification and elevated pH affects the surface potential much less than SCN^– or TPB^– do. These results suggest that the enhancement of Ca²⁺ release by these treatments is not due to a general effect on the membrane surface potential, but rather through the modification of a specific protein. They also suggest that membrane surface charges might play an important role in the control mechanism of Ca²⁺ release.     

Sulfhydryl oxidation and Ca2+ release from sarcoplasmic reticulum

Summary Our interest in the role of sulfhydryl groups (SH) in regulating or altering transport across biological membranes has focused on the significance of a critical SH group associated with the Ca²⁺-release protein from skeletal muscle sarcoplasmic reticulum (SR). We have shown that binding of heavy metals to this group or oxidation of this sulfhydryl to a disulfide induces rapid Ca²⁺ release from SR vesicles [1, 2] and induces contraction in skinned muscle fibers [3]. Several models are described in which oxidation and reduction might control the state of the Ca²⁺-release channel from SR.Abbreviations DTT Dithiothreitol, redox. - oxidation-reduction - SDS Sodium Dodecyl Sulfate - SH Sulfhydryl - SR Sarcoplasmic Reticulum - T-tubule Transverse tubule