Complex effects of ryanodine on the sarcoplasmic reticulum Ca2+ levels in smooth muscle cells

We have studied the effects of ryanodine and inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) with thapsigargin, on both [Ca(2+)](i) and the sarcoplasmic reticulum (SR) Ca(2+) level during caffeine-induced Ca(2+) release in single smooth muscle cells. Incubation with 10 microM ryanodine did not inhibit the first caffeine-induced [Ca(2+)](i) response, although it abolished the [Ca(2+)](i) response to a second application of caffeine. To assess whether ryanodine was inducing a permanent depletion of the internal Ca(2+) stores, we measured the SR Ca(2+) level with Mag-Fura-2. The magnitude of the caffeine-induced reduction in the SR Ca(2+) level was not augmented by incubating cells with 1 microM ryanodine. Moreover, on removal of caffeine, the SR Ca(2+) levels partially recovered in 61% of the cells due to the activity of thapsigargin-sensitive SERCA pumps. Unexpectedly, 10 microM ryanodine instead of inducing complete depletion of SR Ca(2+) stores markedly reduced the caffeine-induced SR Ca(2+) response. It was necessary to previously inhibit SERCA pumps with thapsigargin for ryanodine to be able to induce caffeine-triggered permanent depletion of SR Ca(2+) stores. These data suggest that the effect of ryanodine on smooth muscle SR Ca(2+) stores was markedly affected by the activity of SERCA pumps. Our data highlight the importance of directly measuring SR Ca(2+) levels to determine the effect of ryanodine on the internal Ca(2+) stores.     

Similar effects of cooling and fatigue on eccentric and concentric force-velocity relationships in human muscle.

The purpose of this study was to investigate the effects of muscle temperature and fatigue during stretch (eccentric) and shortening (concentric) contractions of the maximally electrically activated human adductor pollicis muscle. After immersion of the lower arm in water baths of four different temperatures, the calculated muscle temperatures were 36.8, 31.6, 26.6, and 22.3 degrees C. Normalized (isometric force = 100%) eccentric force increased with stretch velocity to maximal values of 136.4 +/- 1.6 and 162.1 +/- 2.0% at 36.8 and 22.3 degrees C, respectively. After repetitive ischemic concentric contractions, fatigue was less at the lower temperatures, and at all temperatures the loss of eccentric force was smaller than the loss of isometric and concentric force. Consequently, normalized eccentric forces increased during fatigue to 159.7 +/- 4.6 and 185.7 +/- 7.3% at 36.8 and 22.3 degrees C, respectively. Maximal normalized eccentric force increased exponentially (r2 = 0.95) when Vmax was reduced by cooling and/or fatiguing contractions. This may indicate that a reduction in cross-bridge cycling rate could underlie the significant increases in normalized eccentric force found with cooling and fatigue.     

Intracellular Cl- fluxes play a novel role in Ca2+ handling in airway smooth muscle

Intracellular Ca(2+) is actively sequestered into the sarcoplasmic reticulum (SR), whereas the release of Ca(2+) from the SR can be triggered by activation of the inositol 1,4,5-trisphosphate and ryanodine receptors. Uptake and release of Ca(2+) across the SR membrane are electrogenic processes; accumulation of positive or negative charge across the SR membrane could electrostatically hinder the movement of Ca(2+) into or out of the SR, respectively. We hypothesized that the movement of intracellular Cl(-) (Cl(i)(-)) across the SR membrane neutralizes the accumulation of charge that accompanies uptake and release of Ca(2+). Thus inhibition of SR Cl(-) fluxes will reduce Ca(2+) sequestration and agonist-induced release. The Cl(-) channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10(-4) M), previously shown to inhibit SR Cl(-) channels, significantly reduced the magnitude of successive acetylcholine-induced contractions of airway smooth muscle (ASM), suggesting a "run down" of sequestered Ca(2+) within the SR. Niflumic acid (10(-4) M), a structurally different Cl(-) channel blocker, had no such effect. Furthermore, NPPB significantly reduced caffeine-induced contraction and increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Depletion of Cl(i)(-), accomplished by bathing ASM strips in Cl(-)-free buffer, significantly reduced the magnitude of successive acetylcholine-induced contractions. In addition, Cl(-) depletion significantly reduced caffeine-induced increases in [Ca(2+)](i). Together these data suggest a novel role for Cl(i)(-) fluxes in Ca(2+) handling in smooth muscle. Because the release of sequestered Ca(2+) is the predominate source of Ca(2+) for contraction of ASM, targeting Cl(i)(-) fluxes may prove useful in the control of ASM hyperresponsiveness associated with asthma.     

Role of the calcium-calpain pathway in cytoskeletal damage after eccentric contractions.

The mechanism(s) underlying eccentric damage to skeletal muscle cytoskeleton remain unclear. We examined the role of Ca(2+) influx and subsequent calpain activation in eccentric damage to cytoskeletal proteins. Eccentric muscle damage was induced by stretching isolated mouse muscles by 20% of the optimal length in a series of 10 tetani. Muscle force and immunostaining of the cytoskeletal proteins desmin, dystrophin, and titin were measured at 5, 15, 30, and 60 min after eccentric contractions and compared with the control group that was subjected to 10 isometric contractions. A Ca(2+)-free solution and leupeptin (100 microM), a calpain inhibitor, were applied to explore the role of Ca(2+) and calpain, respectively, in eccentric muscle damage. After eccentric contractions, decreases in desmin and dystrophin immunostaining were apparent after 5 min that accelerated over the next 60 min. Increased titin immunostaining, thought to indicate damage to titin, was evident 10 min after stretch, and fibronectin entry, indicating membrane disruption, was evident 20 min after stretch. These markers of damage also increased in a time-dependent manner. Muscle force was reduced immediately after stretch and continued to fall, reaching 56 +/- 2% after 60 min. Reducing extracellular calcium to zero or applying leupeptin minimized the changes in immunostaining of cytoskeletal proteins, reduced membrane disruption, and improved the tetanic force. These results suggest that the cytoskeletal damage and membrane disruption were mediated primarily by increased Ca(2+) influx into muscle cells and subsequent activation of calpain.     

Eugenol-induced contractions of saponin-skinned fibers are inhibited by heparin or by a ryanodine receptor blocker

The effects of eugenol on the sarcoplasmic reticulum (SR) and contractile apparatus of chemically skinned skeletal muscle fibers of the frog Rana catesbeiana were investigated. In saponin-skinned fibers, eugenol (5 mmol/L) induced muscle contractions, probably by releasing Ca(2+) from the SR. The Ca(2+)-induced Ca(2+) release blocker ruthenium red (10 micromol/L) inhibited both caffeine- and eugenol-induced muscle contractions. Ryanodine (200 micromol/L), a specific ryanodine receptor/Ca(2+) release channel blocker, promoted complete inhibition of the contractions induced by caffeine, but only partially blocked the contractions induced by eugenol. Heparin (2.5 mg/mL), an inositol 1,4,5-trisphosphate (InsP3) receptor blocker, strongly inhibited the contractions induced by eugenol but had only a small effect on the caffeine-induced contractions. Eugenol neither altered the Ca(2+) sensitivity nor the maximal force in Triton X-100 skinned muscle fibers. These data suggest that muscle contraction induced by eugenol involves at least 2 mechanisms of Ca(2+) release from the SR: one related to the activation of the ryanodine receptors and another through a heparin-sensitive pathway.     

Pathways of Ca²⁺ entry and cytoskeletal damage following eccentric contractions in mouse skeletal muscle

Muscles that are stretched during contraction (eccentric contractions) show deficits in force production and a variety of structural changes, including loss of antibody staining of cytoskeletal proteins. Extracellular Ca(2+) entry and activation of calpains have been proposed as mechanisms involved in these changes. The present study used isolated mouse extensor digitorum longus (EDL) muscles subjected to 10 eccentric contractions and monitored force production, immunostaining of cytoskeletal proteins, and resting stiffness. Possible pathways for Ca(2+) entry were tested with streptomycin (200 μM), a blocker of stretch-activated channels, and with muscles from mice deficient in the transient receptor potential canonical 1 gene (TRPC1 KO), a candidate gene for stretch-activated channels. At 30 min after the eccentric contractions, the isometric force was decreased to 75 ± 3% of initial control and this force loss was reduced by streptomycin but not in the TRPC1 KO. Desmin, titin, and dystrophin all showed patchy loss of immunostaining 30 min after the eccentric contractions, which was substantially reduced by streptomycin and in the TRPC1 KO muscles. Muscles showed a reduction of resting stiffness following eccentric contractions, and this reduction was eliminated by streptomycin and absent in the TRPC1 KO muscles. Calpain activation was determined by the appearance of a lower molecular weight autolysis product and μ-calpain was activated at 30 min, whereas the muscle-specific calpain-3 was not. To test whether the loss of stiffness was caused by titin cleavage, protein gels were used but no significant titin cleavage was detected. These results suggest that Ca(2+) entry following eccentric contractions is through a stretch-activated channel that is blocked by streptomycin and encoded or modulated by TRPC1.     

Hypothermia increases the gain of excitation-contraction coupling in guinea pig ventricular myocytes

Components of excitation-contraction (EC)-coupling were compared at 37 degrees C and 22 degrees C to determine whether hypothermia altered the gain of EC coupling in guinea pig ventricular myocytes. Ca(2+) concentration (fura-2) and cell shortening (edge detector) were measured simultaneously. Hypothermia increased fractional shortening (8.3 +/- 1.7 vs. 2.6 +/- 0.3% at 37 degrees C), Ca(2+) transients (157 +/- 33 vs. 35 +/- 5 nM at 37 degrees C), and diastolic Ca(2+) (100 +/- 9 vs. 60 +/- 6 nM at 37 degrees C) in field-stimulated myocytes (2 Hz). In experiments with high-resistance microelectrodes, the increase in contractions and Ca(2+) transients was accompanied by a twofold increase in action potential duration (APD). When voltage-clamp steps eliminated changes in APD, cooling still increased contractions and Ca(2+) transients. Hypothermia increased sarcoplasmic reticulum (SR) Ca(2+) stores (83 +/- 17 at 37 degrees C to 212 +/- 50 nM, assessed with caffeine) and increased fractional SR Ca(2+) release twofold. In contrast, peak Ca(2+) current was much smaller at 22 degrees C than at 37 degrees C (1.3 +/- 0.4 and 3.5 +/- 0.7 pA/pF, respectively). In cells dialyzed with sodium-free pipette solutions to inhibit Ca(2+) influx via reverse-mode Na(+)/Ca(2+) exchange, hypothermia still increased contractions, Ca(2+) transients, SR stores, and fractional release but decreased the amplitude of Ca(2+) current. The rate of SR Ca(2+) release per unit Ca(2+) current, a measure of EC-coupling gain, was increased sixfold by hypothermia. This increase in gain occurred regardless of whether cells were dialyzed with sodium-free solutions. Thus an increase in EC-coupling gain contributes importantly to positive inotropic effects of hypothermia in the heart.     

Phospholamban knockout increases CaM kinase II activity and intracellular Ca2+ wave activity and alters contractile responses of murine gastric antrum

Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA), and this inhibition is relieved by Ca(2+) calmodulin-dependent protein kinase II (CaM kinase II) phosphorylation. We previously reported significant differences in contractility, SR Ca(2+) release, and CaM kinase II activity in gastric fundus smooth muscles as a result of PLB phosphorylation by CaM kinase II. In this study, we used PLB-knockout (PLB-KO) mice to directly examine the effect of PLB absence on contractility, CaM kinase II activity, and intracellular Ca(2+) waves in gastric antrum smooth muscles. The frequencies and amplitudes of spontaneous phasic contractions were elevated in antrum smooth muscle strips from PLB-KO mice. Bethanecol increased the amplitudes of phasic contractions in antrum smooth muscles from both control and PLB-KO mice. Caffeine decreased and cyclopiazonic acid (CPA) increased the basal tone of antrum smooth muscle strips from PLB-KO mice, but the effects were less pronounced compared with control strips. The CaM kinase II inhibitor KN-93 was less effective at inhibiting caffeine-induced relaxation in antrum smooth muscle strips from PLB-KO mice. CaM kinase II autonomous activity was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. Similarly, the intracellular Ca(2+) wave frequency was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. These findings suggest that PLB is an important modulator of gastric antrum smooth muscle contractility by modulation of SR Ca(2+) release and CaM kinase II activity.     

Physical coupling supports the local Ca2+ transfer between sarcoplasmic reticulum subdomains and the mitochondria in heart muscle

In many cell types, transfer of Ca(2+) released via ryanodine receptors (RyR) to the mitochondrial matrix is locally supported by high [Ca(2+)] microdomains at close contacts between the sarcoplasmic reticulum (SR) and mitochondria. Here we studied whether the close contacts were secured via direct physical coupling in cardiac muscle using isolated rat heart mitochondria (RHMs). "Immuno-organelle chemistry" revealed RyR2 and calsequestrin-positive SR particles associated with mitochondria in both crude and Percoll-purified "heavy" mitochondrial fractions (cRHM and pRHM), to a smaller extent in the latter one. Mitochondria-associated vesicles were also visualized by electron microscopy in the RHMs. Western blot analysis detected greatly reduced presence of SR markers (calsequestrin, SERCA2a, and phospholamban) in pRHM, suggesting that the mitochondria-associated particles represented a small subfraction of the SR. Fluorescence calcium imaging in rhod2-loaded cRHM revealed mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) responses to caffeine-induced Ca(2+) release that were prevented when thapsigargin was added to predeplete the SR or by mitochondrial Ca(2+) uptake inhibitors. Importantly, caffeine failed to increase [Ca(2+)] in the large volume of the incubation medium, suggesting that local Ca(2+) transfer between the SR particles and mitochondria mediated the [Ca(2+)](m) signal. Despite the substantially reduced SR presence, pRHM still displayed a caffeine-induced [Ca(2+)](m) rise comparable with the one recorded in cRHM. Thus, a relatively small fraction of the total SR is physically coupled and transfers Ca(2+) locally to the mitochondria in cardiac muscle. The transferred Ca(2+) stimulates dehydrogenase activity and affects mitochondrial membrane permeabilization, indicating the broad significance of the physical coupling in mitochondrial function.     

Causes of excitation-induced muscle cell damage in isometric contractions: mechanical stress or calcium overload?

Prolonged or unaccustomed exercise leads to muscle cell membrane damage, detectable as release of the intracellular enzyme lactic acid dehydrogenase (LDH). This is correlated to excitation-induced influx of Ca2+, but it cannot be excluded that mechanical stress contributes to the damage. We here explore this question using N-benzyl-p-toluene sulfonamide (BTS), which specifically blocks muscle contraction. Extensor digitorum longus muscles were prepared from 4-wk-old rats and mounted on holders for isometric contractions. Muscles were stimulated intermittently at 40 Hz for 15-60 min or exposed to the Ca2+ ionophore A23187. Electrical stimulation increased 45Ca influx 3-5 fold. This was followed by a progressive release of LDH, which was correlated to the influx of Ca2+. BTS (50 microM) caused a 90% inhibition of contractile force but had no effect on the excitation-induced 45Ca influx. After stimulation, ATP and creatine phosphate levels were higher in BTS-treated muscles, most likely due to the cessation of ATP-utilization for cross-bridge cycling, indicating a better energy status of these muscles. No release of LDH was observed in BTS-treated muscles. However, when exposed to anoxia, electrical stimulation caused a marked increase in LDH release that was not suppressed by BTS but associated with a decrease in the content of ATP. Dynamic passive stretching caused no increase in muscle Ca2+ content and only a minor release of LDH, whereas treatment with A23187 markedly increased LDH release both in control and BTS-treated muscles. In conclusion, after isometric contractions, muscle cell membrane damage depends on Ca2+ influx and energy status and not on mechanical stress.     

O2(*-) production at 37 degrees C plays a critical role in depressing tetanic force of isolated rat and mouse skeletal muscle

To find out whether the decrease in muscle performance of isolated mammalian skeletal muscle associated with the increase in temperature toward physiological levels is related to the increase in muscle superoxide (O(2)(*-)) production, O(2)(*-) released extracellularly by intact isolated rat and mouse extensor digitorum longus (EDL) muscles was measured at 22, 32, and 37 degrees C in Krebs-Ringer solution, and tetanic force was measured in both preparations at 22 and 37 degrees C under the same conditions. The rate of O(2)(*-) production increased marginally when the temperature was increased from 22 to 32 degrees C, but increased fivefold when the temperature was increased from 22 to 37 degrees C in both rat and mouse preparations. This increase was accompanied by a marked decrease in tetanic force after 30 min incubation at 37 degrees C in both rat and mouse EDL muscles. Tetanic force remained largely depressed after return to 22 degrees C for up to 120 min. The specific maximum Ca(2+)-activated force measured in mechanically skinned fibers after the temperature treatment was markedly depressed in mouse fibers but was not significantly depressed in rat muscle fibers. The resting membrane and intracellular action potentials were, however, significantly affected by the temperature treatment in the rat fibers. The effects of the temperature treatment on tetanic force, maximum Ca(2+)-activated force, and membrane potential were largely prevented by 1 mM Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a membrane-permeable superoxide dismutase mimetic, indicating that the increased O(2)(*-) production at physiological temperatures is largely responsible for the observed depression in tetanic force at 37 degrees C by affecting the contractile apparatus and plasma membrane.     

Enhancement of myofilament calcium sensitivity by acute hypoxia in rat distal pulmonary arteries

Hypoxic contraction of pulmonary arterial smooth muscle is thought to require increases in both intracellular Ca(2+) concentration ([Ca(2+)](i)) and myofilament Ca(2+) sensitivity, which may or may not be endothelium-dependent. To examine the effects of hypoxia and endothelium on Ca(2+) sensitivity in pulmonary arterial smooth muscle, we measured the relation between [Ca(2+)](i) and isometric force at 37°C during normoxia (21% O(2)-5% CO(2)) and after 30 min of hypoxia (1% O(2)-5% CO(2)) in endothelium-intact (E+) and -denuded (E-) rat distal intrapulmonary arteries (IPA) permeabilized with staphylococcal α-toxin. Endothelial denudation enhanced Ca(2+) sensitivity during normoxia but did not alter the effects of hypoxia, which shifted the [Ca(2+)](i)-force relation to higher force in E+ and E- IPA. Neither hypoxia nor endothelial denudation altered Ca(2+) sensitivity in mesenteric arteries. In E+ and E- IPA, hypoxic enhancement of Ca(2+) sensitivity was abolished by the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester (30 μM), which shifted normoxic [Ca(2+)](i)-force relations to higher force. In E- IPA, the Rho kinase antagonist Y-27632 (10 μM) shifted the normoxic [Ca(2+)](i)-force relation to lower force but did not alter the effects of hypoxia. These results suggest that acute hypoxia enhanced myofilament Ca(2+) sensitivity in rat IPA by decreasing nitric oxide production and/or activity in smooth muscle, thereby revealing a high basal level of Ca(2+) sensitivity, due in part to Rho kinase, which otherwise did not contribute to Ca(2+) sensitization by hypoxia.     

Cardiac function of two ecologically distinct Neotropical freshwater fish: Curimbata, Prochilodus lineatus (Teleostei, Prochilodontidae), and trahira, Hoplias malabaricus (Teleostei, Erythrinidae)

An isometric muscle preparation was used to investigate the importance of the ventricular sarcoplasmic reticulum (SR) and extracellular Ca(2+) (2.5 up to 10.5 mM) to force generation at 25 degrees C (acclimation temperature) in two ecologically distinct Neotropical teleost fish: Curimbata (active species), and trahira (sedentary species). The post-rest force was studied with and without 10 muM ryanodine in the medium. The positive inotropism observed for both species in response to increases on extracellular Ca(2+) reflected a greater Ca(2+) influx through sarcolemma, as well as an increase in Ca(2+) liberation from the SR by the Ca(2+)-induced Ca(2+) release mechanism. The significant post-rest potentiation recorded for the curimbata and trahira control preparations (3.22+/-0.24 to 6.55+/-0.77 mN mm(-2) and 0.74+/-0.07 to 2.26+/-0.26 mN mm(-2), respectively), was completely inhibited by the addition of ryanodine to the bathing medium, suggesting a potential functionality of SR for both species. Considering the differences in these species habitats, modes of life and levels of activity and the fact of a probable SR Ca(2+) cycling in a physiological temperature, we suggest that the functionality of the SR in these species is probably related to their phylogeny.     

Effects of sevoflurane on the contractility of ferret ventricular myocardium.

Isotonic and isometric variables of contractility and relaxation of isolated ferret right ventricular papillary muscles were measured before and during exposure to incremental concentrations of sevoflurane (0-4.9% vol/vol) (30 degrees C) (n = 9). In a second group of muscles (n = 8), effects of sevoflurane were compared with those of low [Ca(2+)](o) (0.45-2.25 mM in steps of 0.45 mM). Sevoflurane caused a reversible concentration-dependent decrease in contractility (ED(50) of developed force 4.6+/-0.9% vol/vol). When compared with twitches of equal amplitude in low extracellular Ca(2+) concentration, sevoflurane accelerated both isometric and isotonic relaxation. The myocardial depressant effect of sevoflurane is less than that of isoflurane and results mainly from a decrease of intracellular Ca(2+) availability. The abbreviated isometric relaxation likely reflects a decrease in Ca(2+) sensitivity and the faster isotonic relaxation may reflect a mild stimulation of Ca(2+) uptake by the sarcoplasmic reticulum.     

Effects of 4-h ischemia and 1-h reperfusion on rat muscle sarcoplasmic reticulum function

To investigate the hypothesis that ischemia and reperfusion would impair sarcoplasmic reticulum (SR) Ca(2+) regulation in skeletal muscle, Sprague-Dawley rats (n = 20) weighing 290 +/- 3.5 g were randomly assigned to either a control control (CC) group, in which only the effects of anesthetization were studied, or to a group in which the muscles in one hindlimb were made ischemic for 4 h and allowed to recover for 1 h (I). The nonischemic, contralateral muscles served as control (C). Measurements of Ca(2+)-ATPase properties in homogenates and SR vesicles, in mixed gastrocnemius and tibialis anterior muscles, indicated no differences between groups on maximal activity, the Hill coefficient, and Ca(50), defined as the Ca(2+) concentration needed to elicit 50% of maximal activity. In homogenates, Ca(2+) uptake was lower (P < 0.05) by 20-25%, measured at 0.5 and 1.0 microM of free Ca(2+) ([Ca(2+)](f)) in C compared with CC. In SR vesicles, Ca(2+) uptake was lower (P < 0.05) by 30-38% in I compared with CC at [Ca(2+)](f) between 0.5 and 1.5 microM. Silver nitrate induced Ca(2+) release, assessed during both the initial, early rapid (phase 1), and slower, prolonged late (phase 2) phases, in homogenates and SR vesicles, indicated a higher (P < 0.05) release only in phase 1 in SR vesicles in I compared with CC. These results indicate that the alterations in SR Ca(2+) regulation, previously observed after prolonged ischemia by our group, are reversed within 1 h of reperfusion. However, the lower Ca(2+) uptake observed in long-term, nonischemic homogenates suggests that altered regulation may occur in the absence of ischemia.     

Role of FKBP12.6 in hypoxia- and norepinephrine-induced Ca2+ release and contraction in pulmonary artery myocytes

The cellular and molecular processes underlying the regulation of ryanodine receptor (RyR) Ca(2+) release in smooth muscle cells (SMCs) are incompletely understood. Here we show that FKBP12.6 proteins are expressed in pulmonary artery (PA) smooth muscle and associated with type-2 RyRs (RyR2), but not RyR1, RyR3, or IP(3) receptors (IP(3)Rs) in PA sarcoplasmic reticulum. Application of FK506, which binds to FKBPs and dissociates these proteins from RyRs, induced an increase in [Ca(2+)](i) and Ca(2+)-activated Cl(-) and K(+) currents in freshly isolated PASMCs, whereas cyclosporin, an agent known to inhibit calcineurin but not to interact with FKBPs, failed to induce an increase in [Ca(2+)](i). FK506-induced [Ca(2+)](i) increase was completely blocked by the RyR antagonist ruthenium red and ryanodine, but not the IP(3)R antagonist heparin. Hypoxic Ca(2+) response and hypoxic vasoconstriction were significantly enhanced in FKBP12.6 knockout mouse PASMCs. FK506 or rapamycin pretreatment also enhanced hypoxic increase [Ca(2+)](i), but did not alter caffeine-induced Ca(2+) release (SR Ca(2+) content) in PASMCs. Norepinephrine-induced Ca(2+) release and force generation were also markedly enhanced in PASMCs from FKBP12.6 null mice. These findings suggest that FKBP12.6 plays an important role in hypoxia- and neurotransmitter-induced Ca(2+) and contractile responses by regulating the activity of RyRs in PASMCs.     

Effect of endurance exercise on the Ca2+ pumps from transverse tubule and sarcoplasmic reticulum of rabbit skeletal muscle.

The sarcoplasmic reticulum (SR) Ca(2+) pump is the main homeostatic regulatory mechanism in fast skeletal muscle that maintains intracellular Ca(2+) concentration ([Ca(2+)](i)) at the nanomolar level at rest. The transverse tubule (TT) Ca(2+) pump transports cytosolic Ca(2+) to the extracellular space. During prolonged muscular activity, [Ca(2+)](i) may increase. TT and SR isolated microsomal vesicles were highly purified, and the purity was checked by immunoblotting. The present study shows the effects of endurance exercise on the activities and structures of the TT and SR Ca(2+) pumps of fast skeletal muscle from rabbit at rest. The Ca(2+) pump activity increased manifolds in TT but did not change in SR. The protein denaturalization profiles obtained by differential scanning calorimetry showed 1) a shift in the transition temperature and an increase in the enthalpy of the TT Ca(2+) pump and 2) a significant change in the transition temperature of the SR Ca(2+) pump Ca(2+)-binding domain. We conclude that the TT Ca(2+) pump activity was upgraded in association with structural changes to handle the changes in [Ca(2+)](i) and TT lumen Ca(2+) concentration that occur during endurance exercise.     

Ca2+ movements in sarcoplasmic reticulum of rat soleus fibers after hindlimb suspension.

The functional capacity of skeletal muscle sarcoplasmic reticulum (SR) was examined in the slow soleus of rats submitted to 15 days of disuse produced by hindlimb suspension (HS). By using caffeine-induced contractions of single skinned fibers, Ca2+ uptake, Ca2+ release, and passive Ca2+ leakage through the SR membrane were investigated. In the SR of atrophied muscles, the amounts of Ca2+ uptake and Ca2+ release were significantly higher than in the control muscles and were close to those found for a fast muscle, the plantaris. Moreover, the study of the Ca2+ leakage showed that the time required to empty the SR previously loaded with Ca2+ was reduced by a factor of two after HS. Such disturbances of the Ca2+ movements in the SR suggested that alterations of the SR membrane occurred after HS. The results supported the idea that after hindlimb unweighting the slow soleus muscle acquired SR properties that were very much like those of a faster muscle.     

Direct in vivo monitoring of sarcoplasmic reticulum Ca2+ and cytosolic cAMP dynamics in mouse skeletal muscle

Skeletal muscle contraction depends on the release of Ca(2+) from the sarcoplasmic reticulum (SR), but the dynamics of the SR free Ca(2+) concentration ([Ca(2+)](SR)), its modulation by physiological stimuli such as catecholamines, and the concomitant changes in cAMP handling have never been directly determined. We used two-photon microscopy imaging of GFP-based probes expressed in mouse skeletal muscles to monitor, for the first time in a live animal, the dynamics of [Ca(2+)](SR) and cAMP. Our data, which were obtained in highly physiological conditions, suggest that free [Ca(2+)](SR) decreases by approximately 50 microM during single twitches elicited through nerve stimulation. We also demonstrate that cAMP levels rise upon beta-adrenergic stimulation, leading to an increased efficacy of the Ca(2+) release/reuptake cycle during motor nerve stimulation.     

Modification of sarcoplasmic reticulum (SR) Ca2+ release by FK506 induces defective excitation-contraction coupling only when SR Ca2+ recycling is disturbed

This study examined whether the effects of FK506-binding protein dissociation from sarcoplasmic reticulum (SR) Ca(2+) release channels on excitation-contraction (EC) coupling changed when SR Ca(2+) reuptake and (or) the trans-sarcolemmal Ca(2+) extrusion were altered. The steady-state twitch Ca(2+) transient (CaT), cell shortening, post-rest caffeine-induced CaT, and Ca(2+) sparks were measured in rat ventricular myocytes using laser-scanning confocal microscopy. In the normal condition, 50 micromol FK506/L significantly increased steady-state CaT, cell shortening, and post-rest caffeine-induced CaT. When the cells were solely perfused with thapsigargin, FK506 did not reduce any of the states, but when low [Ca(2+)](0) (0.1 mmol/L) was perfused additionally, FK506 reduced CaT and cell shortening, and accelerated the reduction of post-rest caffeine-induced CaT. FK506 significantly increased Ca(2+) spark frequency in the normal condition, whereas it mainly prolonged duration of individual Ca(2+) sparks under the combination of thapsigargin and low [Ca(2+)](0) perfusion. Modification of SR Ca(2+) release by FK506 impaired EC coupling only when released Ca(2+) could not be taken back into the SR and was readily extruded to the extracellular space. Our findings could partly explain the controversy regarding the contribution of FK506-binding protein dissociation to defective EC coupling.