Differential effects of bupivacaine on intracellular Ca2+ regulation: potential mechanisms of its myotoxicity

BACKGROUND: Bupivacaine produces skeletal muscle damage in clinical concentrations. It has been suggested that this may be caused by an increased intracellular level of [Ca2+]. Therefore, the aim of this study was to investigate direct intracellular effects of bupivacaine on Ca2+ release from the sarcoplasmic reticulum (SR), on Ca2+ uptake into the SR, and on Ca2+ sensitivity of the contractile proteins. METHODS: Saponin skinned muscle fibers from the extensor digitorum longus muscle of BALB/c mice were examined according to a standardized procedure described previously. For the assessment of effects on Ca2+ uptake and release from the SR, bupivacaine was added to the loading solution and the release solution, respectively. Force transients and force decays were monitored, and the position of the curve relating relative isometric force free [Ca2+] was evaluated in the presence or absence of bupivacaine. RESULTS: Bupivacaine induces Ca2+ release from the SR. In addition, the Ca2+ loading procedure is suppressed, resulting in smaller caffeine-induced force transients after loading in the presence of bupivacaine. The decay of caffeine-induced force transients is reduced by bupivacaine, and it also shifts [Ca2+]-force relation toward lower [Ca2+]. CONCLUSIONS: These data reveal that bupivacaine does not only induce Ca2+ release from the SR, but also inhibits Ca2+ uptake by the SR, which is mainly regulated by SR Ca2+ adenosine triphosphatase activity. It also has a Ca2+ -sensitizing effect on the contractile proteins. These mechanisms result in increased intracellular [Ca2+] concentrations and may thus contribute to its pronounced skeletal muscle toxicity.     

Comparative effects of bupivacaine and ropivacaine on intracellular calcium transients and tension in ferret ventricular muscle

BACKGROUND: Recent evidence suggests that ropivacaine exerts markedly less cardiotoxicity compared with bupivacaine; however, the mechanisms are not fully understood at the molecular level. METHODS: Isolated ferret ventricular papillary muscles were microinjected with the Ca-binding photoprotein aequorin, and intracellular Ca transients and tension were simultaneously measured during twitch in the absence and presence of bupivacaine or ropivacaine. RESULTS: Bupivacaine and ropivacaine (10, 30, and 100 microm) reduced peak systolic [Ca]i and tension in a concentration-dependent manner. The effects were significantly greater for bupivacaine, particularly on tension (approximately twofold). The percentage reduction of tension was linearly correlated with that of [Ca]i for both anesthetics, with the slope of the relationship being approximately equal to 1.0 for ropivacaine and approximately equal to 1.3 for bupivacaine (slope difference, P < 0.05), suggesting that the cardiodepressant effect of ropivacaine results predominantly from inhibition of Ca transients, whereas bupivacaine suppresses Ca transients and the reaction beyond Ca transients, i.e., myofibrillar activation, as well. BAY K 8644, a Ca channel opener, abolished the inhibitory effects of ropivacaine on Ca transients and tension, whereas BAY K 8644 only partially inhibited the effects of bupivacaine, particularly the effects on tension. CONCLUSION: The cardiodepressant effect of bupivacaine is approximately twofold greater than that of ropivacaine. Bupivacaine suppresses Ca transients more markedly than does ropivacaine and reduces myofibrillar activation, which may at least in part underlie the greater inhibitory effect of bupivacaine on cardiac contractions. These results suggest that ropivacaine has a more favorable profile as a local anesthetic in the clinical settings.     

Effect of ropivacaine on Ca function of skinned skeletal muscle

It is well know that the amide-linked local anesthetics such as lidocaine accelerate Ca induced Ca release (CICR) rate. Since ropivacaine is a new amide-linked local anesthetic, effects of ropivacaine on Ca functions were studied using skinned skeletal muscle. The extensor digitorum longus muscle of male Hartley guinea pigs of about 500 g was prepared for this study. According to Endo's method, CICR rates were measured using chemically skinned fibers. Ropivacaine accelerated the (CICR) rate only at concentrations of 3 mM and 10 mM with pCa 5.0. Ropivacaine at a concentration of 10 mM inhibited initial rate of Ca uptake by sarcoplasmic reticulum. Ca sensitivity of the contractile system was not affected with 10 mM of ropivacaine. These results suggest that ropivacaine can be used safely in patients susceptible to malignant hyperthermia since ropivacaine 3 mM is not a concentration for clinical use.     

EU 4093 decreases intracellular [Ca2+] in skeletal muscle fibers from control and malignant hyperthermia-susceptible swine.

The mechanisms causing the malignant hyperthermia (MH) syndrome are related to a malfunction of intracellular Ca2+ homeostasis and can be prevented or reversed by dantrolene. EU 4093 (Azumolene, 1-[[[5-(4-bromophenyl)-2-oxyzolyl] methylene]amino]-2-4- imidazolidinedione) is a 30-fold more water-soluble analogue of dantrolene that is believed to have the same effects as dantrolene on the intracellular free Ca2+ concentration [( Ca2+]i) in skeletal muscle and that should have similar efficacy in treating and preventing the clinical manifestations of MH in response to a halothane/succinylcholine challenge. To test this hypothesis, experiments were carried out in four controls (Yorkshire) and eight MH-susceptible crossbreed swine (Poland China X Pietrain). The resting [Ca2+]i in normal muscle fibers measured by Ca(2+)-selective microelectrodes was 111 +/- 12 nM (mean +/- standard deviation, n = 30), whereas in the MH muscles the resting [Ca2+]i was 395 +/- 36 nM, (n = 28) (P = 0.0001). EU 4093 decreased [Ca2+]i in MH-susceptible skeletal muscle in a dose-related fashion from 207 to 38 nM after 0.5 to 2.0 mg/kg, respectively, and had a similar effect in control skeletal muscle (58 to 30 nM) after the same doses. In MH-susceptible swine, a dose of 2.0 mg/kg was successful in preventing any clinical signs of the MH syndrome during a subsequent halothane/succinylcholine challenge. A dose of 0.5 mg/kg was able to attenuate but not reverse the clinical signs of the MH syndrome after a halothane challenge, whereas a dose of 1.0 mg/kg was completely successful in reversing this effect in all subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of sevoflurane, isoflurane, and halothane on Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.

BACKGROUND: Although malignant hyperthermia after application of sevoflurane has been reported, little is known about its action on intracellular calcium homeostasis of skeletal muscle. The authors compared the effect of sevoflurane with that of isoflurane and halothane on Ca2+ release of mammalian sarcoplasmic reticulum and applied a novel method to quantify Ca2+ turnover in permeabilized skeletal muscle fibers. METHODS: Liquid sevoflurane, isoflurane, and halothane at 0.6 mM, 3.5 mM, and 7.6 mm were diluted either in weakly calcium buffered solutions with no added Ca2+ (to monitor Ca2+ release) or in strongly Ca2+ buffered solutions with [Ca2+] values between 3 nM and 24.9 microm for [Ca+]-force relations. Measurements were taken on single saponin skinned muscle fiber preparations of BALB/c mice. Individual [Ca2+]force relations were characterized by the Ca2+ concentration at half-maximal force that indicates the sensitivity of the contractile proteins and by the steepness. Each force transient was transformed directly into a Ca2+ transient with respect to the individual [Ca2+]-force relation of the fiber. RESULTS: At 0.6 mM, single force transients induced by sevoflurane were lower compared with equimolar concentrations of isoflurane and halothane (P < 0.05). Similarly, calculated peak Ca2+ transients of sevoflurane were lower than those induced by equimolar halothane (P < 0.05). The Ca2+ concentrations at half maximal force were decreased after the addition of sevoflurane, isoflurane, and halothane in a concentration-dependent manner (P < 0.05). CONCLUSION: Whereas sevoflurane, isoflurane, and halothane similarly increase the Ca2+ sensitivity of the contractile apparatus in skeletal muscle fibers, 0.6 mM sevoflurane induces smaller Ca2+ releases from the sarcoplasmic reticulum than does equimolar halothane.     

Sevoflurane modulation of Ca2+ regulation in skeletal muscle sarcoplasmic reticulum vesicles from young and mature rabbits

Introduction: Developmental differences in splice variants of the two key sarcoplasmic reticulum (SR) calcium regulatory proteins, ryanodine (RyR1), and sarcoendoplasmic reticulum calcium pump (SERCA1) have been linked to various neuromuscular disorders, but not malignant hyperthermia (MH). However, it is unclear whether an age‐related difference in volatile anesthetic‐mediated SR calcium function exists that could add to our current understanding of the clinical presentation of MH syndrome and provide insight into molecular mechanisms for general anesthesia that may have other physiologic and/or pathophysiologic significance. Therefore, the effects of sevoflurane on intracellular calcium regulation in isolated SR membrane vesicles from the skeletal muscle of healthy young rabbits were compared to their adult counterpart using an established in vitro model with the assumption that exposure to sevoflurane would elicit a weaker response in the young SR. Methods: Through dual wavelength spectroscopy of Ca²⁺: Arsenazo III difference absorbance, the effects of sevoflurane on SR Ca²⁺ uptake rate and release in heavy and light fraction SR membrane vesicles isolated from the white muscle of anesthetized, postweaned (age = 6 weeks, n = 5) and adult (age = 6 months, n = 5) male New Zealand rabbits were examined. Results: The adult group showed a 50% increase in Ca²⁺ uptake rate from control at both subclinical and clinically relevant anesthetic concentrations, whereas in the SR from the younger animals, Ca²⁺ uptake rate was not altered by any concentration of sevoflurane. The sensitivity of both the low and high affinity Ca²⁺‐binding sites on RyR1 was increased by sevoflurane to the same extent in the SR vesicles from the young and mature adult rabbits. Interestingly, a greater potency of sevoflurane for the high affinity‐binding site was identified, and this was independent of age. Conclusions: These findings suggest that the sensitivity of the SR to sevoflurane‐mediated Ca²⁺ uptake may be increased with maturity, while an analogous developmental effect on RyR1 is less probable. Nonetheless, this study shows for the first time that a potent inhalational agent such as sevoflurane can influence the high affinity SR calcium‐binding site by lowering the extraluminal concentration of calcium necessary to trigger calcium release. While this may not be of consequence when inhaled anesthetics are administered to normal children or adults, it may have life‐threatening consequences in carriers of RyR1 mutations.     

Effects of sevoflurane on the intracellular Ca2+ transient in ferret cardiac muscle

BACKGROUND: Sevoflurane depresses myocardial contractility by decreasing transsarcolemmal Ca2+ influx. In skinned muscle fibers, sevoflurane affects actin-myosin cross-bridge cycling, which might contribute to the negative inotropic effect. It is uncertain to what extent decreases in Ca2+ sensitivity of the contractile proteins play a role in the negative inotropic effect of sevoflurane in intact cardiac muscle tissue. The aim of this study was to assess whether sevoflurane decreases myofibrillar Ca2+ sensitivity in intact living cardiac fibers and to quantify the relative importance of changes in myofibrillar Ca2+ sensitivity versus changes in myoplasmic Ca2+ availability by sevoflurane. METHODS: The effects of sevoflurane 0-4.05% vol/vol (0-1.5 minimum alveolar concentration [MAC]) on isometric and isotonic variables of contractility and on the intracellular calcium transient were assessed in isolated ferret right ventricular papillary muscles microinjected with the Ca2+-regulated photoprotein aequorin. The intracellular calcium transient was analyzed in the context of a multicompartment model of intracellular Ca2+ buffers in mammalian ventricular myocardium. RESULTS: Sevoflurane decreased contractility, time to peak force, time to half isometric relaxation, and the [Ca2+]i transient in a reversible, concentration-dependent manner. Increasing [Ca2+]o in the presence of sevoflurane to produce peak force equal to control increased intracellular Ca2+ transient higher than control. CONCLUSIONS: Sevoflurane decreases myoplasmic Ca2+ availability and myofibrillar Ca2+ sensitivity in equal proportions except at 4.05% vol/vol (1.5 MAC), where Ca2+ availability is decreased more. These changes are at the basis of the negative inotropic effect of sevoflurane in mammalian ventricular myocardium.     

罗哌卡因和布比卡因对豚鼠心室乳头肌动作电位的影响

目的 观察罗哌卡因和布比卡因对习心室乳头肌纤维动作电位的影响。方法 用含罗哌卡因和布比卡因１、３、５μｇ．ｍｌ＾－１的台氏液分别灌流豚鼠心室乳头肌,记录用药前、用药后及药物洗脱后动作电位各参数的改变：动作电位同值（ＰＡＰ）、动作电位０时相最大上升速度（Ｖｍａｘ）、动作电位５０％复极时程（ＡＰＤ５０）及动作电位９０％复极时程（ＡＰＤ９０）。结果 各浓度局麻药均抑制Ｖｍａｘ,罗哌卡因５μｇ．ｍｌ抑制程     

异丙酚对布比卡因致PC12细胞毒性时细胞Ca2+浓度和一氧化氮合酶活性的影响

目的 评价异丙酚对布比卡因致PC12细胞毒性时细胞Ca2+浓度和一氧化氮合酶(NOS)活性的影响.方法 PC12细胞悬液(105/ml)随机分成4组:对照组(C组)、异丙酚组(P组)、布比卡因组(B组)和异丙酚+布比卡因组(PB组),每组PC12细胞分别接种于36孔板(每孔1 ml,每组9孔)、激光共聚焦显微镜专用培养皿(每皿1 ml,每组6皿)和24孔板(每孔 1 ml,每组6孔).C组加入D-Hank液500 μl;P组加入异丙酚至终浓度为2 mmol/L;B组加入布比卡因至终浓度为0.09 mmol/L;PB组同时加入异丙酚和布比卡因,终浓度分别为2 mmol/L和0.09 mmol/L.于36孔板中孵育24 h后测定PC12细胞凋亡率;于激光共聚焦显微镜专用培养皿中孵育6、24 h时,测定PC12细胞游离Ca2+浓度;于24孔板中孵育6、24 h时测定细胞NOS活性.结果 与C组相比,B组和PB组PC12细胞游离Ca2+浓度、NOS活性和凋亡率均升高(P<0.01),P组上述指标差异无统计学意义(P>0.05);与B组相比,PB组PC12细胞游离Ca2+浓度、NOS活性和凋亡率均降低(P<0.05).结论 在细胞水平,异祆丙酚可能通过抑制NOS活性和钙超载,减轻布比卡因诱导的神经毒性.     

Effects of halothane and isoflurane on the intracellular Ca2+ transient in ferret cardiac muscle

BACKGROUND: Halothane and isoflurane depress myocardial contractility by decreasing transsarcolemmal Ca2+ influx and Ca2+ release from the sarcoplasmic reticulum. Decreases in Ca2+ sensitivity of the contractile proteins have been shown in skinned cardiac fibers, but the relative importance of this effect in intact living myocardium is unknown. The aims of this study were to assess whether halothane and isoflurane decrease myofibrillar Ca2+ sensitivity in intact, living cardiac fibers and to quantify the relative importance of changes in myofibrillar Ca2+ sensitivity versus changes in myoplasmic Ca2+ availability caused by these anesthetics. METHODS: The effects of halothane and isoflurane (0-1.5 times the minimum alveolar concentration (MAC) in three equal increments) on isometric and isotonic variables of contractility and on the intracellular calcium transient were assessed in isolated ferret right ventricular papillary muscle microinjected with the Ca2+-regulated photoprotein aequorin. The intracellular calcium transient was analyzed in the context of a multicompartment model of intracellular Ca2+ buffers in mammalian ventricular myocardium. RESULTS: Halothane and isoflurane decreased contractility, time-to-peak force, time to half-isometric relaxation, and intracellular Ca2+ transient in a reversible, concentration-dependent manner. Halothane, but not isoflurane, slowed the increase and the decrease of the intracellular Ca2+ transient. Increasing extracellular Ca2+ in the presence of anesthetic to produce peak force equal to control values increased intracellular Ca2+ to values higher than control values. CONCLUSIONS: Halothane decreases myoplasmic Ca2+ availability more than isoflurane; halothane and isoflurane decrease myofibrillar Ca2+ sensitivity to the same extent; in halothane at 0.5 MAC and isoflurane at 1.0 MAC, the decrease in Ca2+ sensitivity is already fully apparent; halothane decreases intracellular Ca2+ availability more than myofibrillar Ca2+ sensitivity; and isoflurane decreases myoplasmic Ca2+ availability and Ca2+ sensitivity to the same extent, except at 1.5 times the MAC, which decreases Ca2+ availability more.     

Ca2+ uptake and Ca2+ release by skeletal muscle sarcoplasmic reticulum: differing sensitivity to inhalational anesthetics

The effects of halothane, enflurane, and isoflurane were measured on two different mechanisms of Ca2+ regulation by isolated skeletal muscle sarcoplasmic reticulum (SR) membranes. A 100,000-dalton Ca2+-ATPase protein transports Ca2+ from outside to inside the SR membrane. At concentration ranges representing anesthetic levels of 0.06 to 2.3 times MAC, halothane, enflurane, and isoflurane each increased rate of Ca2+ uptake by SR. Each concentration of isoflurane produced a greater rate of Ca2+ uptake, whereas halothane and enflurane produced maximum stimulation of Ca2+ uptake at 1 and 1.6 times MAC, respectively. The second Ca2+ regulation mechanism studied was a Ca2+ release channel in the SR membrane. The release of Ca2+ via this mechanism requires a critical threshold Ca2+ load (nmol Ca2+/mg SR protein) for Ca2+-induced Ca2+ release to occur. Each anesthetic tested effectively lowered the critical Ca2+ load threshold for Ca2+ release, i.e., the Ca2+ channel was more readily induced to an open state in the presence of anesthetic. The concentrations of anesthetics having this effect on the putative Ca2+ channel were between 0.0026 and 0.078 MAC equivalents for each agent, and these concentrations are much lower than the anesthetic concentrations affecting Ca2+ uptake. These data show that in isolated skeletal muscle SR membranes a Ca2+ channel release function is altered at anesthetic concentrations far below those that change Ca2+ uptake function by a Ca2+-ATPase and below concentrations of the volatile agents producing clinical anesthesia. The Ca2+ channel effect may represent protein-anesthetic interaction, whereas the Ca2+-ATPase effect may occur by a generalized SR membrane perturbation by the anesthetics.     

The role of Ca2+ influx and intracellular Ca2+ release in the muscarinic-mediated contraction of mammalian urinary bladder smooth muscle

OBJECTIVE To study the involvement of extracellular Ca2+ and the properties of the intracellular Ca2+ ([Ca2+]i) stores on the carbachol-induced contraction of mammalian urinary bladder smooth muscle strips under polarized and depolarized conditions. MATERIALS AND METHODS: Strips of bladder were suspended between platinum ring electrodes in a cylindrical organ bath (0.2 mL) and continuously superfused with Krebs' solution at 1 mL/min. The effect of nifedipine, cyclopiazonic acid (CPA), thapsigargin, procaine, ryanodine and caffeine before and during a 10-s application of 100 microm carbachol under polarized conditions were studied. The effect of these drugs was also assessed under depolarized conditions using a protocol that allowed a more detailed assessment of the role of [Ca2+]i stores, consisting of emptying the stores by exposure to Ca2+-free solution, rapidly refilling them by a 10-s application of 81.5 mm Ca2+ (priming), returning to the Ca2+-free solution for 3 min and then applying 100 microm carbachol (10 s) in Ca2+-free solution (store release). RESULTS: Under polarized conditions, nifedipine and Ca2+ removal almost completely inhibited the carbachol-induced contractions. CPA increased the amplitude and duration of both carbachol- and electrical field stimulation-induced contractions. Although ryanodine had no inhibitory effect, caffeine and procaine significantly inhibited the carbachol-induced contraction. Under depolarized conditions nifedipine blocked both priming and store release contractions. CPA, thapsigargin, procaine and ryanodine significantly increased the priming and inhibited the store release contractions. However, caffeine virtually abolished both priming and store release contractions. CONCLUSION: These results suggest that in guinea-pig urinary bladder smooth muscle the Ca2+ necessary for contraction enters the cell through voltage-dependent dihydropyridine-sensitive Ca2+ channels and is pumped into an intracellular store that is released by carbachol. Under polarized conditions, the blockade of sarco-endoplasmic reticulum calcium ATP-ase (SERCA) with CPA increases [Ca2+]i and carbachol-induced contractions. The effects of caffeine and procaine suggest that store release involves ryanodine receptors and calcium-induced calcium release. Under depolarized conditions, Ca2+ entry is blocked by nifedipine and the stores diminish. Stored Ca2+ is also greatly reduced by the blockade of SERCA with either CPA or thapsigargin. Procaine, ryanodine and caffeine blocked the store release contractions, suggesting that this involves ryanodine receptors and calcium-induced calcium release.     

Inhibitory effects of halo thane on high K+–induced canine tracheal smooth muscle contraction and intracellular Ca 2+ increment

Halothane is a potent bronchodilator. The effects of halothane on isolated canine tracheal smooth muscle contraction and intracellular Ca²⁺ increment induced by a high concentration of K⁺ were investigated to clarify how this anaesthetic decreases intracellular Ca²⁺ concentration ([Ca²⁺]i), an important second messenger. The tension of the muscle strips was measured using an isometric transducer, and [Ca²⁺]i was measured using a surface fluorescence spectroscopy. Exposure to a 72.7 mM K⁺ solution increased muscle tension and [Ca²⁺]i. Halothane (1,2,3 and 4% at the vaporiser) was introduced by bubbling in the presence of the 72.7 mM K⁺, and significantly decreased both this elevated muscle tension and the [Ca²⁺]i in a concentration–dependent manner. Similarly, slowly decreasing concentrations of K⁺ (48.5, 36.4, 24.2 and 18.2 mM) significantly decreased both of these variables. There was no significant difference between the slopes of the two regression lines of changes in muscle tension and changes in [Ca²⁺]i. From the evidence that tonic increase in [Ca²⁺]i by high concentrations of K⁺ is due to the influx of Ca²⁺ via L–type voltage channels, halothane may modify the L–type channels to decrease Ca²⁺ influx. In conclusion, halothane inhibits the high K⁺ –induced canine airway smooth muscle contraction by decreasing [Ca²⁺]i. The decrease in [Ca²⁺]i by halothane might be due to inhibition of voltage–operated channels, especially the L–type channels.     

Isoflurane neuroprotection in rat hippocampal slices decreases with aging: changes in intracellular Ca2+ regulation and N-methyl-D-aspartate receptor-mediated Ca2+ influx

BACKGROUND: Most in vitro neuroprotection studies with isoflurane have involved cells obtained during the embryonic or early postnatal period. However, in mature rodents, isoflurane neuroprotection does not persist. The authors determined whether neuroprotection of hippocampal slices with isoflurane decreases with aging and is due to decreased intracellular Ca regulation and survival protein phosphorylation. METHODS: Hippocampal slices from 5-day-old, 1-month-old, and 19- to 23-month-old rats were deprived of oxygen and glucose for 5-30 min in media bubbled with 1% isoflurane. Cell death was assessed in the CA1, CA3, and dentate regions, and intracellular Ca concentration was measured in CA1 neurons. N-methyl-d-aspartate receptor (NMDAR)-dependent Ca influx was measured and the phosphorylation of NMDARs, and the survival proteins Akt and mitogen-activated protein kinase p42/44 were quantified. RESULTS: Twenty minutes of oxygen and glucose deprivation killed approximately 40-60% of neurons in CA3 and dentate in all age groups. Isoflurane, 1%, reduced death of CA1, CA3, and dentate neurons in slices from 5-day-old rats but not those from 23-month-old rats. In 5-day slices, isoflurane attenuated NMDAR-mediated Ca influx, whereas in aging slices, Ca influx was increased protein kinase C. In aging slices, isoflurane did not increase the phosphorylation of Akt and p42/44. CONCLUSIONS: Isoflurane neuroprotection of hippocampal slices during oxygen and glucose deprivation decreases with age. Isoflurane does not prevent large increases in intracellular Ca concentration during oxygen and glucose deprivation and does not induce the phosphorylation of the prosurvival proteins in aging slices. A protein kinase C-mediated increase in NMDAR activity may result in increased excitotoxicity and decreased neuroprotection by volatile anesthetics in the aging brain.     

The role of Ca2+ influx for insulin-mediated glucose uptake in skeletal muscle

The involvement of Ca(2+) in insulin-mediated glucose uptake is uncertain. We measured Ca(2+) influx (as Mn(2+) quenching or Ba(2+) influx) and 2-deoxyglucose (2-DG) uptake in single muscle fibers isolated from limbs of adult mice; 2-DG uptake was also measured in isolated whole muscles. Exposure to insulin increased the Ca(2+) influx in single muscle cells. Ca(2+) influx in the presence of insulin was decreased by 2-aminoethoxydiphenyl borate (2-APB) and increased by the membrane-permeable diacylglycerol analog 1-oleyl-2-acetyl-sn-glycerol (OAG), agents frequently used to block and activate, respectively, nonselective cation channels. Maneuvers that decreased Ca(2+) influx in the presence of insulin also decreased 2-DG uptake, whereas increased Ca(2+) influx was associated with increased insulin-mediated glucose uptake in isolated single cells and whole muscles from both normal and insulin-resistant obese ob/ob mice. 2-APB and OAG affected neither basal nor hypoxia- or contraction-mediated 2-DG uptake. 2-APB did not inhibit the insulin-mediated activation of protein kinase B or extracellular signal-related kinase 1/2 in whole muscles. In conclusion, alterations in Ca(2+) influx specifically modulate insulin-mediated glucose uptake in both normal and insulin-resistant skeletal muscle. Moreover, the present results indicate that Ca(2+) acts late in the insulin signaling pathway, for instance, in the GLUT4 translocation to the plasma membrane.     

Effect of superoxide dismutase plus catalase on Ca2+ transport in ischemic and reperfused skeletal muscle

Cytotoxic oxygen metabolites may contribute to skeletal muscle damage associated with ischemia and reperfusion. This study utilized a rat hindlimb ischemia model to investigate the effect of pretreatment with oxygen free radical scavengers superoxide dismutase (SOD) and catalase (CAT) on skeletal muscle Ca2+ uptake by sarcoplasmic reticulum (SR) in limbs subjected to periods of ischemia and reperfusion. SOD and CAT were conjugated to polyethylene glycol to prolong their half lives. Anesthetized rats (ca. 350 g) received an iv injection of either conjugated SOD (2 mg/kg) plus CAT (3.5 mg/kg) (n = 6, Treated Group) or 0.9 saline (4 ml/kg) (n = 6, Control Group) 5 min before unilateral hindlimb tourniquet ischemia of 3 hr duration. After 19 hr of reperfusion, muscle from each lower leg was excised and homogenized. Skeletal muscle SR was isolated by differential centrifugation. ATP-dependent Ca2+ uptake by the SR was then measured with dual wavelength spectrophotometry and used as an index of muscle function. Pretreatment with SOD and CAT maintained higher rates of Ca2+ uptake by SR of skeletal muscle from postischemic reperfused limbs (Treated Group 2.29 +/- 0.21 vs Control Group, 1.61 +/- 0.06 mumole Ca2+/mg protein/min). These results implicate cytotoxic oxygen metabolites in the pathogenesis of ischemic reperfusion skeletal muscle injury.     

Interaction of bupivacaine and tetracaine with the sarcoplasmic reticulum Ca2+ release channel of skeletal and cardiac muscles

BACKGROUND: Although various local anesthetics can cause histologic damage to skeletal muscle when injected intramuscularly, bupivacaine appears to have an exceptionally high rate of myotoxicity. Research has suggested that an effect of bupivacaine on sarcoplasmic reticulum Ca2+ release is involved in its myotoxicity, but direct evidence is lacking. Furthermore, it is not known whether the toxicity depends on the unique chemical characteristics of bupivacaine and whether the toxicity is found only in skeletal muscle. METHODS: The authors studied the effects of bupivacaine and the similarly lipid-soluble local anesthetic, tetracaine, on the Ca2+ release channel-ryanodine receptor of sarcoplasmic reticulum in swine skeletal and cardiac muscle. [3H]Ryanodine binding was used to measure the activity of the Ca2+ release channel-ryanodine receptors in microsomes of both muscles. RESULTS: Bupivacaine enhanced (by two times at 5 mM) and inhibited (66% inhibition at 10 mM) [3H]ryanodine binding to skeletal muscle microsomes. In contrast, only inhibitory effects were observed with cardiac microsomes (about 3 mM for half-maximal inhibition). Tetracaine, which inhibits [3H]ryanodine binding to skeletal muscle microsomes, also inhibited [3H]ryanodine binding to cardiac muscle microsomes (half-maximal inhibition at 99 microM). CONCLUSIONS: Bupivacaine's ability to enhance Ca2+ release channel-ryanodine receptor activity of skeletal muscle sarcoplasmic reticulum most likely contributes to the myotoxicity of this local anesthetic. Thus, the pronounced myotoxicity of bupivacaine may be the result of this specific effect on Ca2+ release channel-ryanodine receptor superimposed on a nonspecific action on lipid bilayers to increase the Ca2+ permeability of sarcoplasmic reticulum membranes, an effect shared by all local anesthetics. The specific action of tetracaine to inhibit Ca2+ release channel-ryanodine receptor activity may in part counterbalance the nonspecific action, resulting in moderate myotoxicity.     

Role of intracellular Ca2+ stores in smooth muscle of human penile erectile tissue

Objective: In human erectile tissue smooth muscle contraction and detumescence are highly dependent on an increase in cytosolic [Ca²⁺]. The Ca²⁺ influx can be derived from the extracellular space or from intracellular sarcoplasmic stores. The role of both pathways was evaluated in an organ bath study on human cavernosal strips. Patients and methods: The tissue was obtained from 12 patients with chronic erectile dysfunction. The effects of Ca²⁺-free solution, ryanodine, caffeine and of nifedipine on electrically and adrenergically induced contractions were evaluated. Results: Following an incubation period of 10 min in Ca²⁺-free solution the electrically induced contraction was reduced to 20%, whereas the contraction induced by phenylephrine (PE) was only reduced to 64 ± 6% (mean ± SEM). Ryanodine inhibited the PE-contraction to 30 ± 6% and the additional application of caffeine or nifedipine further reduced the contraction to 11% and 8%. Conclusion: The results give evidence for a role of intracellular Ca²⁺-stores in human cavernosal tissue. Whether the more marked effect of ryanodine in tissue from patients with erectile failure in comparison with similar experiments in rabbit cavernosal tissue might be a sign of an increased cavernosal contractility in these patients remains to be shown in future experiments with normal erectile tissue. Received: 4 March 1997 / Accepted: 10 November 1997     

Differential effects of halothane, enflurane, and isoflurane on Ca2+ transients and papillary muscle tension in guinea pigs.

These studies were designed to examine the effects of inhalational anesthetics on rapid changes in myocardial intracellular Ca2+ and Ca2+ sensitivity of the contractile apparatus. The effects of halothane, enflurane, and isoflurane on rapid changes in intracellular Ca2+ (Ca2+ transients as measured with bioluminescent protein aequorin) and contractile characteristics were compared in guinea pig right ventricular papillary muscles. In addition to examination of their potencies at equianesthetic concentrations, the effects of these agents on alterations in Ca2+ sensitivity at myofilaments were also investigated. The negative inotropic effects of halothane (0.65 and 1.15%) and enflurane (1.0 and 2.2%) were dose-dependent and closely related to a decrease in Ca2+ transients. In the presence of isoflurane (0.77 and 1.6%), the contractile force decreased in a dose-dependent manner, but the decrease was significantly less as compared to that with equianesthetic concentrations of halothane and enflurane. An additional feature observed in the presence of isoflurane was a dissociation between intracellular Ca2+ availability and contractile force. Although the magnitude of the Ca2+ transients did not change when the percentage of isoflurane was increased from 0.77 to 1.6, the contractile force decreased. Because of these findings, the effects of halothane (1.2%), enflurane (2.2%), and isoflurane (1.6%) on the relationship between intracellular Ca2+ and tension developed in the papillary muscle were examined in order to assess myofibrillar responsiveness to Ca2+. The results indicate that only isoflurane slightly but significantly shifted the Ca2+/isometric tension curve toward higher intracellular Ca2+ concentrations; no differences were observed in the absence and presence of equianesthetic concentrations of halothane and enflurane.(ABSTRACT TRUNCATED AT 250 WORDS)