Depression Of Ca2 Research Articles

Effects of high-intensity interval exercise on muscle fatigue and SR function in rats: a comparison with moderate-intensity continuous exercise.

In this study, we compared muscle fatigue induced by high-intensity interval exercise (HIIE) and moderate-intensity continuous exercise (MICE), with a focus on changes in the function of sarcoplasmic reticulum (SR) and myofibril. To achieve the aim of this study with mechanically skinned fibers with sealed transverse tubules and intact SR membrane, myofibrillar Ca2+ sensitivity, depolarization-induced force, and action potential-induced force were evaluated. Rat gastrocnemius muscles were subjected to HIIE-mimicking or MICE-mimicking stimulation in situ. The number of contractions was the same for MICE- and HIIE-mimicking stimulation (total of 360 contractions). Three hours after cessation of stimulation, the superficial regions of gastrocnemius muscles were dissected and used for biochemical and skinned fiber analyses. At 3 h of recovery, forces at 20 and 100 Hz in whole muscles had returned to resting levels in MICE but not HIIE muscles. The reduced glutathione content was decreased only in HIIE muscles. Both MICE- and HIIE-mimicking stimulation resulted in an increase in myofibrillar Ca2+ sensitivity in skinned fibers. Only HIIE-mimicking stimulation led to a decrease in the ratio of force at 1 Hz to that at 50 Hz and the ratio of depolarization-induced force to the maximum Ca2+-activated force. These results reflect the properties of type IIX and IIB fibers (the latter is not expressed in human skeletal muscles) and suggest that HIIE requires longer recovery periods than those normally used with MICE, which is ascribable to long-lasting depressions in SR Ca2+ release.NEW & NOTEWORTHY Over the past decade, high-intensity interval exercise (HIIE) training has received attention as a more efficient training to improve endurance capacity. It is unclear, however, whether the extent of acute exercise-related muscle fatigue differs between HIIE and moderate-intensity continuous exercise, traditional endurance training. Here we provide evidence that restoration of force production takes a longer time after HIIE, which is ascribable to long-lasting depressions in Ca2+ release of the sarcoplasmic reticulum.

Acidosis decreases the Ca2+ sensitivity of thin filaments by preventing the first actomyosin interaction.

Intracellular acidosis is a putative agent of skeletal muscle fatigue, in part, because it depresses the calcium (Ca2+) sensitivity of the myofilaments. However, the molecular mechanism behind this depression in Ca2+ sensitivity is unknown, providing a significant challenge to a complete understanding of the fatigue process. To elucidate this mechanism, we directly determined the effect of acidosis on the ability of a single myosin molecule to bind to a regulated actin filament in a laser trap assay. Decreasing pH from 7.4 to 6.5 significantly (P < 0.05) reduced the frequency of single actomyosin-binding events at submaximal (pCa 8-pCa 6) but not at maximal Ca2+ concentration (pCa 5-pCa 4). To delineate whether this was due to a direct effect on myosin versus an indirect effect on the regulatory proteins troponin (Tn) and tropomyosin (Tm), binding frequency was also quantified in the absence of Tn and Tm. This revealed that acidosis did not significantly alter the frequency of actomyosin binding events in the absence of regulatory proteins (1.4 ± 0.15 vs. 1.4 ± 0.15 events/s for pH 7.4 and 6.5, respectively). Acidosis also did not significantly affect the size of myosin's powerstroke or the duration of binding events in the presence of regulatory proteins, at every [Ca2+]. These data suggest acidosis impedes activation of the thin filament by competitively inhibiting Ca2+ binding to TnC. This slows the rate at which myosin initially attaches to actin; therefore, less cross bridges will be bound and generating force at any given submaximal [Ca2+]. These data provide a molecular explanation for the acidosis-induced decrease in force observed at the submaximal Ca2+ concentrations that might contribute to the loss of force during muscle fatigue.

Ryanodine Receptor 1 mRNA Expression is Increased by Post‐Natal Maturation and Long Term Hypoxia in Sheep Pulmonary Arteries

Ryanodine receptors (RyRs) are well regarded for being important to vasorelaxation that occurs during the birth transition and with hypoxic pulmonary vasoconstrictor responses. Previous studies from our group show that RyR mediated Ca2+ spark events are suppressed in fetal pulmonary arterial myocytes and that these events are significantly modified by exposure to long term high altitude hypoxia. To begin to resolve the molecular underpinnings of these physiological responses we performed quantitative real time PCR for mRNA expression of the RyR1 isoform, which is one of three relevant isoforms that are likely to be expressed in pulmonary arterial myocytes. Pulmonary arterial (PA) segments were isolated from near‐term fetal or adult sheep housed at low altitude (300m) or exposed to high altitude (3,801m) for over 100 days. Comparisons of RyR1 mRNA expression were then made to the housekeeping gene beta‐actin. Based on these preliminary studies we found that the ΔCt for RyR1 mRNA expression relative to beta actin was significantly lower in the fetus relative to the adult independent of altitude. The ΔΔCt corresponded to a 12–17 fold increase in RyR1 mRNA expression due to maturation, which potentially accounts for previously shown maturational related enhancements in Ca2+ spark activity. Altitude caused a 2.8 fold increase in RyR1 mRNA expression in adult PA that correlate with previously shown maturational induced increases in spark activity. Interestingly, a 3.8 fold increase in RyR1 mRNA expression observed in fetal PA following hypoxia did not correspond to the hypoxia‐related depression in Ca2+ spark activity that we previously reported. Overall, these preliminary studies show pronounced changes in RyR1 mRNA expression that are likely to be important drivers of pulmonary vascular development as well as underlie pathologies due to long term high altitude exposure.Support or Funding InformationThis work is supported by The National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development grant number HD083132 and HD069746.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Volatile Anesthetic Depression of Ca2+ Entry Into and Glutamate Release from Cultured Cerebellar Granule Neurons

Background: Volatile anesthetics (VAs) are known to have actions on a variety of ligand- and voltage gated ion channels, and thereby inhibit neuronal function. VA effects mediated by actions on voltage-gated Ca channels (VGCCs) were determined by studying their effects on the depolarization-induced rise in intracellular Ca2+ transients and the consequent glutamate release in cultured neonatal rat cerebellar granule neurons. Methods: Using a glutamate dehydrogenase-coupled assay for glutamate release, and fura-2 to measure intracellular [Ca2+] ([Ca2+]i), neurons at 37℃ were depolarized by a rapid increase in [K+]o from 5 to 55 mM. Actions of halothane, isoflurane, enflurane, and sevoflurane were compared with effects of altered [Mg2+]o, and by specific blockade of L-, P/Q- and/or N-type VGCC by nicardipine, ω-agatoxin IVA, and ω-conotoxin-GVIA, respectively. Whole-cell patch-clamp studies in these same neurons of VGCC Ba2+ currents were also performed at 22℃. Results: Clinical VA concentrations dose-dependently depressed both peak [Ca2+]i and glutamate release by 35-70%. With N- and/or L-type VGCC blockade, VAs caused a further marked decrease in [Ca2+]i transients. VAs depressed whole cell patch-clamped Ba2+ currents in these granule cell neurons by 35-40%. Conclusions: VAs depress Ca2+ entry by inhibiting a variety of VGCCs, and thereby reduce neuronal glutamate release. This action may contribute to the mechanism of anesthesia as well as provide protection during ischemic insults that cause neuronal injury. Citation: Carl Lynch III, Ning Miao, Kaoru Nagao, Joseph J. Pancrazio. Volatile anesthetic depression of Ca2+ entry into and glutamate release from cultured cerebellar granule neurons. J Anesth Perioper Med 2017; 4: 45-59. doi:10.24015/JAPM.2017.0016This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

T-Type voltage-sensitive calcium channels mediate mechanically-induced intracellular calcium oscillations in osteocytes by regulating endoplasmic reticulum calcium dynamics

One of the earliest responses of bone cells to mechanical stimuli is a rise in intracellular calcium (Ca2+), and osteocytes in particular exhibit robust oscillations in Ca2+ when subjected to loading. Previous studies implicate roles for both the endoplasmic reticulum (ER) and T-Type voltage-sensitive calcium channels (VSCC) in these responses, but their interactions or relative contributions have not been studied. By observing Ca2+ dynamics in the cytosol (Ca2+cyt) and the ER (Ca2+ER), the focus of this study was to explore the role of the ER and T-Type channels in Ca2+ signaling in bone cells. We demonstrate that inhibition of T-Type VSCC in osteocytes significantly reduces the number of Ca2+cyt responses and affects Ca2+ER depletion dynamics. Simultaneous observation of Ca2+ exchange among these spaces revealed high synchrony between rises in Ca2+cyt and depressions in Ca2+ER, and this synchrony was significantly reduced by challenging T-Type VSCC. We further confirmed that this effect was mediated directly through the ER and not through store-operated Ca2+ entry (SOCE) pathways. Taken together, our data suggests that T-Type VSCC facilitate the recovery of Ca2+ER in osteocytes to sustain mechanically-induced Ca2+ oscillations, uncovering a new mechanism underlying the behavior of osteocytes as mechanosensors.

Enhancement of Voltage-Gated K+ Channels and Depression of Voltage-Gated Ca2+ Channels Are Involved in Quercetin-Induced Vasorelaxation in Rat Coronary Artery

Quercetin is one of the most common flavonoids in the human daily diet. Its affects the coronary artery, especially L-type voltage-gated Ca2+ channels and voltage-gated K+ channels in the arterial smooth muscle cells, which are poorly understood. The present experiments were designed to study the myogenic effect of quercetin and its possible underlying mechanisms in the rat coronary artery. A wire myograph was used to observe the myogenic effects. Arterial smooth muscle cells were freshly isolated from the rat coronary artery and the intracellular free Ca2+ concentration was measured with molecular probe fluo-4-AM. The effects of quercetin on L-type voltage-gated Ca2+ channels and voltage-gated K+ channels were studied using a whole-cell patch clamp. Quercetin (3-30 µM) produced a depression and relaxation on the contraction induced by KCl or the thromboxane A2 analog 9,11-Dideoxy-9α,11α-methanoepoxy prostaglandin F 2α . The vasorelaxation was attenuated by 4-aminopyridine, a specific voltage-gated K+ channel inhibitor, but was not affected by the NG-nitro-L-arginine methylester ester (a nitric oxide synthesis inhibitor), glibenclamide (a ATP-activated K+ channel inhibitor), iberiotoxin (a Ca2+-activated K+ channel inhibitor), BaCl2 (an inward rectifier K+ channel inhibitor), or by endothelium denudation. At the same concentrations, quercetin reduced the KCl-induced elevation of the intracellular free Ca2+ concentration, inhibited the inward Ca2+ currents through L-type voltage-gated Ca2+ channels, and increased the outward K+ currents through voltage-gated K+ channels in the rat coronary artery smooth muscle cells. Collectively, our results demonstrate that quercetin possesses vasospasmolytic effects in RCA and suggest that depression of the Ca2+ influx through L-type voltage-gated Ca2+ channels and augmentation of voltage-gated K+ channel activity in the myocytes may underlie coronary relaxation.

Abstract 275: Berbamine Confers Cardioprotection Against Ischemia-Reperfusion Injury by Attenuating Ca 2+ Overload and Preventing Calpain Activation Through Protection of Mitochondria

Reperfusion is an essential treatment to salvage ischemia myocardium from necrosis. However, the reperfusion can also lead to additional damage. Ischemic preconditioning is a powerful way to initiate intrinsic adaptive responses protecting the heart from subsequent severe ischemia/reperfusion (I/R) injury that can be mimicked by pharmacological preconditioning. However, so far few agents are clinically available for patients with ischemia heart disease. There is a growing interest in exploring effective cardioprotective components extracted from Traditional Chinese Medicine because they have been practiced for thousands of years, providing a vast source of pharmaceutical materials. Berbamine, a kind of bisbenzylisochinoline alkaloids derived from the roots, barks and stems of Barberry , was reported to protect myocardium from ischemia/reperfusion (I/R) injury, but the underlying mechanisms are largely unknown. Here, we investigated whether berbamine confers cardioprotection via preventing calpain activation and protein degradation. We identified here that berbamine pretreatment from 10 to 100 nM concentration-dependently improved post-ischemic myocardial function in isolated Sprague-Dawley rat hearts subjected to 30 min ischemia/45 min reperfusion. Similar protection was confirmed in isolated cardiomyocytes characterized by the attenuation of I/R-induced intracellular Ca 2+ overloading and the depression of Ca 2+ transients and cell shortening. Moreover, I/R-induced decreases of Ca 2+ handling proteins were significantly attenuated by berbamine. Further analysis showed that those protections were related to the suppression of I/R-induced calpain activity and the protection of mitochondrial function by berbamine. These findings suggest that berbamine confers cardioprotections against I/R injury by attenuating Ca 2+ overloading and preventing calpain activation; and the protection of mitochondria is critical for the cardioprotection.

The Changes of [Ca2+] in Sr and Ca2+ Release Flux during Fatiguing Activation of Mouse Skeletal Muscle Fibers

Sustained activity leads to muscle fatigue, cellular correlates of which include depression in Ca2+ released per action potential (Gyorke, JPhysiol, 1993; Westerblad, JAppPhysiol, 1993). This decrease is also observed in cells activated with voltage clamp pulse trains (Royer, JGP, 2010), which points at effectors downstream from the voltage sensor, including depletion of SR Ca2+ (Allen, JAppPhysiol, 2011). In mouse FDB cells expressing the biosensor D4cpv-calsequestrin loaded with X-rhod1-AM or in mice constitutively expressing the troponin-based FRET biosensor TnXX (Mank, NatureM, 2008) we measure [Ca2+]SR, [Ca2+]cyto and release flux. Fatiguing activation is produced by 0.5s trains of action potentials at 2s intervals. Concentrations were measured before the tetanus (rest) or at its end (tet) in the rested condition (t0), upon 1 or 3 min of stimulation (t1,t3) and after 10 min recovery (A10). Flux was calculated as amount released/tetanus duration. Further analysis of this quantitative picture of Ca2+ handling during fatigue indicates that the reduction in Ca2+ release is the outcome of interconnected decreases in SR load, Ca2+ release permeability and SR Ca2+ buffering power.Funded by NIAMS and EU Framework7 Program.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Transient Ca2+ depletion of the sarcoplasmic reticulum at the onset of reperfusion

Myocardial stunning is a contractile dysfunction that occurs after a brief ischaemic insult. Substantial evidence supports that this dysfunction is triggered by Ca2+ overload during reperfusion. The aim of the present manuscript is to define the origin of this Ca2+ increase in the intact heart. To address this issue, Langendorff-perfused mouse hearts positioned on a pulsed local field fluorescence microscope and loaded with fluorescent dyes Rhod-2, Mag-fluo-4, and Di-8-ANEPPS, to assess cytosolic Ca2+, sarcoplasmic reticulum (SR) Ca2+, and transmembrane action potentials (AP), respectively, in the epicardial layer of the hearts, were submitted to 12 min of global ischaemia followed by reperfusion. Ischaemia increased cytosolic Ca2+ in association with a decrease in intracellular Ca2+ transients and a depression of Ca2+ transient kinetics, i.e. the rise time and decay time constant of Ca2+ transients were significantly prolonged. Reperfusion produced a transient increase in cytosolic Ca2+ (Ca2+ bump), which was temporally associated with a decrease in SR-Ca2+ content, as a mirror-like image. Caffeine pulses (20 mM) confirmed that SR-Ca2+ content was greatly diminished at the onset of reflow. The SR-Ca2+ decrease was associated with a decrease in Ca2+ transient amplitude and a shortening of AP duration mainly due to a decrease in phase 2. To the best of our knowledge, this is the first study in which SR-Ca2+ transients are recorded in the intact heart, revealing a previously unknown participation of SR on cytosolic Ca2+ overload upon reperfusion in the intact beating heart. Additionally, the associated shortening of phase 2 of the AP may provide a clue to explain early reperfusion arrhythmias.

Depression of Ca2+/calmodulin‐dependent protein kinase II in dorsal root ganglion neurons after spinal nerve ligation

The enzyme calcium/calmodulin-dependent protein kinase II (CaMKII) is associated with memory and its alpha isoform is critical for development of activity-induced synaptic changes. Therefore, we hypothesized that CaMKII is involved in altered function of dorsal root ganglion (DRG) neurons after neuronal injury. To test this hypothesis, Sprague-Dawley rats were made hyperalgesic by L5 and L6 spinal nerve ligation (SNL), and changes in total phosphorylated and unphosphorylated CaMKII (tCaMKII) and phosphorylated form of its alpha isoform (pCaMKIIalpha) were analyzed using immunochemistry in different subpopulations of DRG. SNL did not induce any changes in tCaMKII between experimental groups, while the overall percentage of pCaMKIIalpha-positive neurons in injured L5 DRG SNL (24.8%) decreased significantly when compared to control (41.7%). SNL did not change the percentage of pCaMKIIalpha/N52 colabeled neurons but decreased the percentage of N52-negative nonmyelinated neurons that expressed pCaMKIIalpha from 27% in control animals to 11% after axotomy. We also observed a significant decrease in the percentage of small nonpeptidergic neurons labeled with IB4 (37.6% in control vs. 4.0% in L5 SNL DRG), as well as a decrease in the percentage of pCaMKIIalpha/IB4 colabeled neurons in injured L5 DRGs (27% in control vs. 1% in L5 DRG of SNL group). Our results show that reduction in pCaMKIIalpha levels following peripheral injury is due to the loss of IB4-positive neurons. These results indicate that diminished afferent activity after axotomy may lead to decreased phosphorylation of CaMKIIalpha.

Cardiac oxidative stress determination and myocardial morphology after a single ecstasy (MDMA) administration in a rat model

Experimental and clinical data indicate that 3,4-methylenedioxy-N-methylamphetamine (MDMA) abuse can produce significant cardiovascular toxicity. A mechanism may be a direct toxic effect of redox active metabolites of MDMA. To evaluate the effect of a single MDMA dose on cellular antioxidant defence system and to investigate the morphology in male albino rats, total glutathione (GSH), oxidised glutathione (GSSG), ascorbic acid (AA), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and malondialdehyde (MDAL) were studied. The effects were evaluated at 3, 6, 16 and 24 h after MDMA administration. Antioxidant enzymes activity was significantly reduced: GPx (-24%) and SOD (-50%) after 3 h and GR (-19%) after 6 h from treatment. AA levels decrease (-37%) after 3 h and (-30%) after 6 h; MDAL level increased (+119%) after 3 h; GSH levels decreased after 3 (31.3%) and 6 h (37.9%) from MDMA treatment. GSSG content was not affected by ecstasy administration. Myocardial contraction band necrosis (CBN) was already visible in rats killed at 6 h. After 16 h, macrophagic monocytes around the necrotic myocardial cells were observed, and within 24 h, this infiltrate became more widespread with an early removal of the necrotic material. Calcium deposits were observed within ventricular cardiomyocytes with intact nuclei and sarcomeres. Single administration of MDMA can significantly alter the cellular antioxidant defence system and produce oxidative stress which may result in lipid peroxidation and disruption of Ca(2 +) homeostasis. The depression in Ca(2+) regulatory mechanism by reactive oxygen species ultimately results in intracellular Ca(2 +) overload, CBN and cell death.

Negative Inotropy of the Gastric Proton Pump Inhibitor Pantoprazole in Myocardium From Humans and Rabbits

Proton pump inhibitors are used extensively for acid-related gastrointestinal diseases. Their effect on cardiac contractility has not been assessed directly. Under physiological conditions (37 degrees C, pH 7.35, 1.25 mmol/L Ca2+), there was a dose-dependent decrease in contractile force in ventricular trabeculae isolated from end-stage failing human hearts superfused with pantoprazole. The concentration leading to 50% maximal response was 17.3+/-1.3 microg/mL. Similar observations were made in trabeculae from human atria, normal rabbit ventricles, and isolated rabbit ventricular myocytes. Real-time polymerase chain reaction demonstrated the expression of gastric H+/K+-adenosine triphosphatase in human and rabbit myocardium. However, measurements with BCECF-loaded rabbit trabeculae did not reveal any significant pantoprazole-dependent changes of pH(i). Ca2+ transients recorded from field-stimulated fluo 3-loaded myocytes (F/F0) were significantly depressed by 10.4+/-2.1% at 40 microg/mL. Intracellular Ca2+ fluxes were assessed in fura 2-loaded, voltage-clamped rabbit ventricular myocytes. Pantoprazole (40 microg/mL) caused an increase in diastolic [Ca2+]i by 33+/-12%, but peak systolic [Ca2+]i was unchanged, resulting in a decreased Ca2+ transient amplitude by 25+/-8%. The amplitude of the L-type Ca2+ current (I(Ca,L)) was reduced by 35+/-5%, and sarcoplasmic reticulum Ca2+ content was reduced by 18+/-6%. Measurements of oxalate-supported sarcoplasmic reticulum Ca2+ uptake in permeabilized cardiomyocytes indicated that pantoprazole decreased Ca2+ sensitivity (Kd) of sarcoplasmic reticulum Ca2+ adenosine triphosphatase: control, Kd=358+/-15 nmol/L; 40 microg/mL pantoprazole, Kd=395+/-12 nmol/L (P<0.05). Pantoprazole also acted on cardiac myofilaments to reduced Ca2+-activated force. Pantoprazole depresses cardiac contractility in vitro by depression of Ca2+ signaling and myofilament activity. In view of the extensive use of this agent, the effects should be evaluated in vivo.

Sex differences affect Ca2+ sparks parameters in normal and diabetic rat ventricular cardiomyoctes

The present study was designed to determine whether the properties of local Ca2+ releases and its related regulatory mechanisms might provide insight into the role of sex differences in heart functions of normal and diabetic female rats. Cells were obtained by enzymatic dispersion of hearts from normal and diabetic (STZ; 50 mg/kg) rats. Basal [Ca2+]i as well as Ca2+ sparks frequency of cardiomyoctes from the diabetic rats significantly increased with respect to control rats. Ca2+ transients exhibited significantly reduced amplitude, and prolonged time courses as well as depressed Ca2+ loading of SR in diabetics. Spatio-temporal properties of the Ca2+ sparks in cardiomyocytes isolated from diabetic rats were also significantly altered to being almost parallel to the changes of the Ca2+ transients. In addition, cardiac RyR2 from diabetic hearts were hyperphosphorylated and protein levels of both RyR2 and FK506 binding protein 12.6 (FKBP12.6) depleted. All these data were similar to the males data published previously except less hyperphosphorylation level of RyR2 and less increase in basal [Ca2+]i as well as less depression in Ca2+ loading of SR compared to the diabetic males. Our data related with higher levels of RyR2 and FKBP12.6 in normal females describe some of the mechanisms that may underlie gender specific differences, especially a female advantage in the development of cardiac disease. Supported by TUBITAKSBAG-3056, SBAG-PIA-10 and DPT 98K-121670.

Nonlinear [Ca2+] Signaling in Dendrites and Spines Caused by Activity-Dependent Depression of Ca2+Extrusion

Spine Ca2+ triggers the induction of synaptic plasticity and other adaptive neuronal responses. The amplitude and time course of Ca2+ signals specify the activation of the signaling pathways that trigger different forms of plasticity such as long-term potentiation and depression. The shapes of Ca2+ signals are determined by the dynamics of Ca2+ sources, Ca2+ buffers, and Ca2+ extrusion mechanisms. Here we show in rat CA1 pyramidal neurons that plasma membrane Ca2+ pumps (PMCAs) and Na+/Ca2+ exchangers are the major Ca2+ extrusion pathways in spines and small dendrites. Surprisingly, we found that Ca2+ extrusion via PMCA and Na+/Ca2+ exchangers slows in an activity-dependent manner, mediated by intracellular Na+ and Ca2+ accumulations. This activity-dependent depression of Ca2+ extrusion is, in part, attributable to Ca2+-dependent inactivation of PMCAs. Ca2+ extrusion recovers from depression with a time constant of 0.5 s. Depression of Ca2+ extrusion provides a positive feedback loop, converting small differences in stimuli into large differences in Ca2+ concentration. Depression of Ca2+ extrusion produces Ca2+ concentration dynamics that depend on the history of neuronal activity and therefore likely modulates the induction of synaptic plasticity.

Oxidative stress and septic shock: metabolic aspects of oxygen-derived free radicals generated in the liver during endotoxemia

This review describes the role of oxidative stress caused by endotoxin challenge in sepsis or septic shock symptoms. We observed that endotoxin injection resulted in lipid peroxide formation and membrane damage (near 60-150 kDa) in the livers of experimental animals, causing decreased levels of scavengers or quenchers of free radicals. The administration of alpha-tocopherol completely prevented injury to the liver plasma membrane caused by endotoxin, and suggested that lipid peroxidation by free radicals might occur in a tissue ischemic state, probably by disseminated intravascular coagulation (DIC), in endotoxemia. In mice, depression of Ca(2+)-ATPase activity in the liver plasma membrane may contribute to the membrane damage caused by endotoxin, and the increase of [Ca(2+)](i) in the liver cytoplasm may partially explain the oxidative stress that occurs in endotoxemia. It seems that endotoxin-induced free radical formation is regulated by Ca(2+) mobilization. Moreover, we have suggested that the oxidative stress caused by endotoxin may be due, at least in part, to the changes in endogenous zinc or selenium regulation during endotoxemia. Interestingly, the extent of TNF-alpha-induced oxidative stress may be the result of a synergism between TNF-alpha and gut-derived endotoxin. It is likely that bacterial or endotoxin translocation plays a significant role in TNF-alpha-induced septic shock. On the other hand, although nitric oxide (NO) has been implicated in the pathogenesis of vascular hyporesponsiveness and hypotension in septic shock in our experimental model, it is unlikely that NO plays a significant role in liver injury caused by free radical generation in endotoxemia.

Role of dual-site phospholamban phosphorylation in intermittent hypoxia-induced cardioprotection against ischemia-reperfusion injury

Cardioprotection by intermittent high-altitude (IHA) hypoxia against ischemia-reperfusion (I/R) injury is associated with Ca(2+) overload reduction. Phospholamban (PLB) phosphorylation relieves cardiac sarcoplasmic reticulum (SR) Ca(2+)-pump ATPase, a critical regulator in intracellular Ca(2+) cycling, from inhibition. To test the hypothesis that IHA hypoxia increases PLB phosphorylation and that such an effect plays a role in cardioprotection, we compared the time-dependent changes in the PLB phosphorylation at Ser(16) (PKA site) and Thr(17) (CaMKII site) in perfused normoxic rat hearts with those in IHA hypoxic rat hearts submitted to 30-min ischemia (I30) followed by 30-min reperfusion (R30). IHA hypoxia improved postischemic contractile recovery, reduced the maximum extent of ischemic contracture, and attenuated I/R-induced depression in Ca(2+)-pump ATPase activity. Although the PLB protein levels remained constant during I/R in both groups, Ser(16) phosphorylation increased at I30 and 1 min of reperfusion (R1) but decreased at R30 in normoxic hearts. IHA hypoxia upregulated the increase further at I30 and R1. Thr(17) phosphorylation decreased at I30, R1, and R30 in normoxic hearts, but IHA hypoxia attenuated the depression at R1 and R30. Moreover, PKA inhibitor H89 abolished IHA hypoxia-induced increase in Ser(16) phosphorylation, Ca(2+)-pump ATPase activity, and the recovery of cardiac performance after ischemia. CaMKII inhibitor KN-93 also abolished the beneficial effects of IHA hypoxia on Thr(17) phosphorylation, Ca(2+)-pump ATPase activity, and the postischemic contractile recovery. These findings indicate that IHA hypoxia mitigates I/R-induced depression in SR Ca(2+)-pump ATPase activity by upregulating dual-site PLB phosphorylation, which may consequently contribute to IHA hypoxia-induced cardioprotection against I/R injury.

Effects of protein kinase C dependent phosphorylation and a familial hypertrophic cardiomyopathy-related mutation of cardiac troponin I on structural transition of troponin C and myofilament activation.

In experiments reported here, we compared tension and thin filament Ca(2+) signaling in preparations containing either wild-type cardiac troponin I (cTnI) or a mutant cTnI with an R146G mutation [cTnI(146G)] linked to familial hypertrophic cardiomyopathy. Myofilament function is altered in association with cTnI phosphorylation by protein kinase C (PKC), which is activated in hypertrophy. Whether there are differential effects of PKC phosphorylation on cTnI compared to cTnI(146G) remains unknown. We therefore also studied cTnI and cTnI(146G) with PKC sites mutated to Glu, which mimics phosphorylation. Compared to cTnI controls, binary complexes with either cTnI(146G) or cTnI(43E/45E/144E) had a small effect on Ca(2+)-dependent structural opening of the N-terminal regulatory domain of cTnC as measured using Förster resonance energy transfer. However, this structural change was significantly reduced in the cTnC-cTnI(43E/45E/144E/146G) complex. Exchange of cTnI in skinned fiber bundles with cTnI(146G) induced enhanced Ca(2+) sensitivity and an elevated resting tension. Exchange of cTnI with cTnI(43E/45E/144E) induced a depression in Ca(2+) sensitivity and maximum tension. However, compared to cTnI(146G), cTnI(43E/45E/144E/146G) had little additional effects on myofilament response to Ca(2+). By comparing activation of tension to the open state of the N-domain of cTnC with variations in the state of cTnI, we were able to provide data supporting the hypothesis that activation of cardiac myofilaments is tightly coupled to the open state of the N-domain of cTnC. Our data also support the hypothesis that pathological effects of phosphorylation are influenced by mutations in cTnI.

Direct myocardial depressant effect of naloxone: mechanical and electrophysiological actions in vitro.

Naloxone, an opioid antagonist, has a cardiovascular pressor effect and has been used in various types of shock states. The aim was to determine the non-opioid effect on direct cardiac muscle contractility and explore the mechanism using guinea pig right ventricular papillary muscles. With institutional approval, isometric contractile force was measured in modified normal and 26 mM K+ Tyrode solution at various stimulation rates. The effects of naloxone on sarcoplasmic recticulum function were evaluated by measuring rested-state contraction in low Na+ (25 mM) Tyrode solution and rapid cooling contracture in modified normal Tyrode solution. Normal and slow action potentials (APs) were measured using conventional microelectrode technique. Patch clamp study was performed to examine the direct effect on Ca2+ current in cardiac ventricular myocytes. Naloxone (50, 100, and 200 microM) caused a concentration-dependent depression of peak force and maximal rate of force development. Modest depression, approximately 20%, was shown in rested-state contraction in low Na+ Tyrode solution. Naloxone (100 microM) modestly depressed the rapid cooling contracture to 80 +/- 3% of baseline, accompanied by prolongation of time to peak contracture by approximately 37%. In 26 mM K+ Tyrode solution, naloxone (100 microM) markedly and selectively depressed the late force development. While naloxone (100 microM) did not alter the amplitude and dV/dt-max in normal and slow APs at 0.25 Hz, AP duration was prolonged significantly. In patch clamp experiment, naloxone (50 microM) depressed Ca2+ current by approximately 50%. The direct myocardial depressant effect of naloxone appeared to be in part caused by depression of Ca2+ influx through cardiac membrane. Sarcoplasmic reticulum function appeared to be modestly depressed.

Direct myocardial depressant effect of methylmethacrylate monomer: mechanical and electrophysiologic actions in vitro.

The present study explored the mechanism of direct myocardial depression by methylmethacrylate monomer (MMA). Isometric contraction of isolated guinea pig right ventricular papillary muscle was measured in modified normal and 26 mm K+ Tyrode solutions at various stimulation rates. Normal and slow action potentials were evaluated by conventional microelectrode technique. MMA effects on various aspects of sarcoplasmic reticulum function were evaluated by its effect on rapid-cooling contractures, rested-state contraction in rat papillary muscle in modified normal Tyrode solution, and in guinea pig papillary muscle under low Na+ (25 mm) Tyrode solution. Whole cell patch clamp techniques were applied to measure the inward Ca2+ currents (I(Ca)). MMA (0.5, 1.5, and 4.7 mm) caused concentration-dependent depression of peak force and maximal rate of force development to approximately 70, 50, and 20% of baseline from rested state to 3 Hz stimulation rates, respectively. Depression of peak force and maximal rate of force development by MMA was dependent on stimulation frequency, with less depression at higher stimulation rates. In low Na+ Tyrode solution, 1.5 mm MMA depressed peak force of rat and guinea pig myocardium by 20-30%. In 26 mm K+ Tyrode solution, 0.5 and 1.5 mm MMA caused selective and marked concentration-dependent depression of late force development (0.5 mm: approximately 60% of baseline, 1.5 mm: approximately 30% of baseline) with no alteration in early force development. MMA (1.5 mm) depressed rapid-cooling contracture to 53 +/- 10% of baseline, accompanied by approximately 63% prolongation of time to peak contracture. In patch clamp studies, MMA reduced I(Ca) in a concentration-dependent manner. The direct myocardial depressant effect of MMA seems to be caused in part by depression of Ca2+ influx through cardiac membrane, while depolarization-activated sarcoplasmic reticulum Ca2+ release appears modestly depressed.

Down-regulation of Voltage-gated Ca2+ Channels by Neuronal Calcium Sensor-1 Is β Subunit-specific

Neuronal Ca(2+) sensor protein-1 (NCS-1) is a member of the Ca(2+) binding protein family, with three functional Ca(2+) binding EF-hands and an N-terminal myristoylation site. NCS-1 is expressed in brain and heart during embryonic and postnatal development. In neurons, NCS-1 facilitates neurotransmitter release, but both inhibition and facilitation of the Ca(2+) current amplitude have been reported. In heart, NCS-1 co-immunoprecipitates with K(+) channels and modulates their activity, but the potential effects of NCS-1 on cardiac Ca(2+) channels have not been investigated. To directly assess the effect of NCS-1 on the various types of Ca(2+) channels we have co-expressed NCS-1 in Xenopus oocytes, with Ca(V)1.2, Ca(V)2.1, and Ca(V)2.2 Ca(2+) channels, using various subunit combinations. The major effect of NCS-1 was to decrease Ca(2+) current amplitude, recorded with the three different types of alpha(1) subunit. When expressed with Ca(V)2.1, the depression of Ca(2+) current amplitude induced by NCS-1 was dependent upon the identity of the beta subunit expressed, with no block recorded without beta subunit or with the beta(3) subunit. Current-voltage and inactivation curves were also slightly modified and displayed a different specificity toward the beta subunits. Taken together, these data suggest that NCS-1 is able to modulate cardiac and neuronal voltage-gated Ca(2+) channels in a beta subunit specific manner.