Furrow-related contractions are inhibited but furrow-unrelated contractions are not affected in af mutant eggs of Xenopus laevis

In embryos from af mutant females of Xenopus laevis, the cleavage furrows stayed on the surface and cytoplasmic divisions did not take place at all, while nuclear divisions continued (Kubota et al., 1991). To gain insights into the roles of the normal product of af on early development, contractile events which have been observed in the period from fertilization until first cleavage in wild-type eggs were examined in af mutant eggs. Activation waves, activation contraction, and surface contraction waves which were identical to those in wild-type eggs were observed in af eggs by time-lapse video recording. However, second polar body elimination was inhibited in af eggs, although a sign of the polar body formation was indicated by the cytoplasmic bulge of the egg surface as seen by light and electron microscopy. These results indicate that the normal product of af regulates furrow-related contractile events which involve formation of the contractile ring, but exerts no effects on furrow-unrelated contractions in early Xenopus eggs.     

Reversible block of the calcium release channel/ryanodine receptor by protamine, a heparin antidote

Channel activity of the calcium release channel from skeletal muscle, ryanodine receptor type 1, was measured in the presence and absence of protamine sulfate on the cytoplasmic side of the channel. Single-channel activity was measured after incorporating channels into planar lipid bilayers. Optimally and suboptimally calcium-activated calcium release channels were inactivated by the application of protamine to the cytoplasmic side of the channel. Recovery of channel activity was not observed while protamine was present. The addition of protamine bound to agarose beads did not change channel activity, implying that the mechanism of action involves an interaction with the ryanodine receptor rather than changes in the bulk calcium concentration of the medium. The block of channel activity by protamine could be reversed either by removal by perfusion with buffer or by the addition of heparin to the cytoplasmic side of the channel. Microinjection of protamine into differentiated C(2)C(12) mouse muscle cells prevented caffeine-induced intracellular calcium release. The results suggest that protamine acts on the ryanodine receptor in a similar but opposite manner from heparin and that protamine can be used as a potent, reversible inhibitor of ryanodine receptor activity.     

Apoptotic pathways are inhibited by leptin receptor activation in neutrophils

Leptin regulates food intake as well as metabolic, endocrine, and immune functions. It exerts proliferative and antiapoptotic activities in a variety of cell types, including T cells. Leptin also stimulates macrophages and neutrophils, and its production is increased during inflammation. In this study, we demonstrate that human neutrophils express leptin surface receptors under in vitro and in vivo conditions, and that leptin delays apoptosis of mature neutrophils in vitro. The antiapoptotic effects of leptin were concentration dependent and blocked by an anti-leptin receptor mAb. The efficacy of leptin to block neutrophil apoptosis was similar to G-CSF. Using pharmacological inhibitors, we obtained evidence that leptin initiates a signaling cascade involving PI3K- and MAPK-dependent pathways in neutrophils. Moreover, leptin delayed the cleavage of Bid and Bax, the mitochondrial release of cytochrome c and second mitochondria-derived activator of caspase, as well as the activation of both caspase-8 and caspase-3 in these cells. Taken together, leptin is a survival cytokine for human neutrophils, a finding with potential pathologic relevance in inflammatory diseases.     

Mitochondrial respiratory parameters in cardiac tissue: a novel method of assessment by using saponin-skinned fibers

Respiratory parameters of cardiac mitochondria were determined in the bundles of cardiac fibers skinned by using saponin that specifically removed sarcolemma, but left intracellular structures intact. In the assay medium which simulated the ion composition of cardiac cytoplasm maximal value of state 3 oxygen consumption per mol cytochromes aa3 was close to that value for isolated mitochondria. Ischemia and isopreterenol treatment were found to affect respiratory parameters of mitochondria in saponin-skinned fibers, among them creatine-stimulated respiration decreased most significantly, (3-4)-times under these conditions. The method described can be easily applied for determination of the mitochondrial respiratory parameters in small (5-10 mg) biopsy samples from human heart.     

Dihydropyridine and ryanodine receptor binding after eccentric contractions in mouse skeletal muscle.

The purpose of this study was to determine whether there are alterations in the dihydropyridine and/or ryanodine receptors that might explain the excitation-contraction uncoupling associated with eccentric contraction-induced skeletal muscle injury. The left anterior crural muscles (i.e., tibialis anterior, extensor digitorum longus, and extensor hallucis longus) of mice were injured in vivo by 150 eccentric contractions. Peak isometric tetanic torque of the anterior crural muscles was reduced approximately 45% immediately and 3 days after the eccentric contractions. Partial restoration of peak isometric tetanic and subtetanic forces of injured extensor digitorum longus muscles by 10 mM caffeine indicated the presence of excitation-contraction uncoupling. Scatchard analysis of [3H]ryanodine binding indicated that the number of ryanodine receptor binding sites was not altered immediately postinjury but decreased 16% 3 days later. Dihydropyridine receptor binding sites increased approximately 20% immediately after and were elevated to the same extent 3 days after the injury protocol. Muscle injury did not alter the sensitivity of either receptor. These data suggest that a loss or altered sensitivity of the dihydropyridine and ryanodine receptors does not contribute to the excitation-contraction uncoupling immediately after contraction-induced muscle injury. We also concluded that the loss in ryanodine receptors 3 days after injury is not the primary cause of excitation-contraction uncoupling at that time.     

Excitation-contraction coupling in skeletal muscle fibers injected with the InsP3 blocker, heparin

Heparin, an inhibitor of inositol trisphosphate (InsP3)-induced Ca2+ release in smooth muscle and non-muscle cells, was injected into intact frog skeletal muscle fibres. Ca2+ release from the sarcoplasmic reticulum was elicited by the normal action potential mechanism and monitored by both fura-2 fluorescence and an intrinsic birefringence signal. Both optical signals, and hence Ca2+ release, were unaffected by high concentrations of heparin. This result argues against a major physiological role of InsP3 as a chemical messenger of excitation-contraction coupling in skeletal muscle.     

Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

We studied cation regulation of wild-type ryanodine receptor type 1 (_WTRyR1), type 3 (_WTRyR3), and RyR3/RyR1 chimeras (Ch) expressed in 1B5 dyspedic myotubes. Using [³H]ryanodine binding to sarcoplasmic reticulum (SR) membranes, Ca²⁺ titrations with _WTRyR3 and three chimeras show biphasic activation that is allosterically coupled to an attenuated inhibition relative to _WTRyR1. Chimeras show biphasic Mg²⁺ inhibition profiles at 3 and 10 μM Ca²⁺, no observable inhibition at 20 μM Ca²⁺ and monophasic inhibition at 100 μM Ca²⁺. Ca²⁺ imaging of intact myotubes expressing Ch-4 exhibit caffeine-induced Ca²⁺ transients with inhibition kinetics that are significantly slower than those expressing _WTRyR1 or _WTRyR3. Four new aspects of RyR regulation are evident: (1) high affinity (H) activation and low affinity (L) inhibition sites are allosterically coupled, (2) Ca²⁺ facilitates removal of the inherent Mg²⁺ block, (3) _WTRyR3 exhibits reduced cooperativity between H activation sites when compared to _WTRyR1, and (4) uncoupling of these sites in Ch-4 results in decreased rates of inactivation of caffeine-induced Ca²⁺ transients.     

Cardiac ryanodine receptor is absent in type I slow skeletal muscle fibers: immunochemical and ryanodine binding studies

Cardiac ryanodine receptor was purified from canine ventricle as a single polypeptide of Mr 400,000 by a stepwise sucrose density gradient centrifugation and heparin-Sepharose CL-4B column chromatography. The [3H]ryanodine binding capacity (Bmax) was 60-fold enriched from cardiac microsomes without a change in affinity for [3H]ryanodine. The purity of the final preparation was determined to be greater than 95% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using this purified preparation as an antigen, we produced six monoclonal antibodies which immunoprecipitated the cardiac ryanodine receptor. Three of these antibodies recognized the cardiac receptor on immunoblot analysis. In contrast, no protein in the microsomes isolated from Type I (slow) or Type II (fast) skeletal muscles was recognized by these antibodies. The [3H]ryanodine binding to cardiac and skeletal muscle microsomes was dependent on free Ca2+ concentration. In skeletal muscle microsomes, the [3H]ryanodine binding was remarkably enhanced by the addition of ATP or KCl and inhibited by high free Ca2+, whereas it was less sensitive to these agents in cardiac microsomes. All of these results clearly demonstrate that the cardiac ryanodine receptor is different from the skeletal muscle receptors and is not present even in Type I (slow) skeletal muscle fibers, in which cardiac isoforms of some of the muscle proteins are constitutively expressed.     

Ca2+对骨骼肌钙释放通道的调节

钙释放通道（calcium release channel）又称Ryanodine受体（RyR）,是细胞内质网膜上介导细胞内钙信号转导的离子通道。RyR1在骨骼肌细胞的兴奋-收缩偶联过程中起重要作用,是肌质网快速释放Ca^2＋的通道。许多调节因素,如一些内源性蛋白（FK结合蛋白、钙调素、钙结合蛋白）和一些离子（Ca^2＋、Mg^2＋）,通过不同的作用位点与RyR1结合,调控RyR1的结构与功能。研究表明,Ca^2＋是众多调节RyR1因素中的核心成分和前提条件,其对RyR1的结构与功能有重要的调控作用。     

Branched fibers in dystrophic mdx muscle are associated with a loss of force following lengthening contractions

We demonstrated that the susceptibility of skeletal muscle to injury from lengthening contractions in the dystrophin-deficient mdx mouse is directly linked with the extent of fiber branching within the muscles and that both parameters increase as the mdx animal ages. We subjected isolated extensor digitorum longus muscles to a lengthening contraction protocol of 15% strain and measured the resulting drop in force production (force deficit). We also examined the morphology of individual muscle fibers. In mdx mice 1–2 mo of age, 17% of muscle fibers were branched, and the force deficit of 7% was not significantly different from that of age-matched littermate controls. In mdx mice 6–7 mo of age, 89% of muscle fibers were branched, and the force deficit of 58% was significantly higher than the 25% force deficit of age-matched littermate controls. These data demonstrated an association between the extent of branching and the greater vulnerability to contraction-induced injury in the older fast-twitch dystrophic muscle. Our findings demonstrate that fiber branching may play a role in the pathogenesis of muscular dystrophy in mdx mice, and this could affect the interpretation of previous studies involving lengthening contractions in this animal. skeletal muscle; mdx mouse; lengthening contraction; Duchenne muscular dystrophy     

Effects of an alpha-helical ryanodine receptor C-terminal tail peptide on ryanodine receptor activity: modulation by Homer

We have determined the structure of a domain peptide corresponding to the extreme 19 C-terminal residues of the ryanodine receptor Ca2+ release channel. We examined functional interactions between the peptide and the channel, in the absence and in the presence of the regulatory protein Homer. The peptide was partly alpha-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels. The peptide (0.1-10 microM) inhibited skeletal ryanodine receptor channels when the cytoplasmic Ca2+ concentration was 1 microM; but with 10 microM cytoplasmic Ca2+, skeletal ryanodine receptors were activated by < or = 1.0 microM peptide and inhibited by 10 microM peptide. Cardiac ryanodine receptors on the other hand were inhibited by all peptide concentrations, at both Ca2+ concentrations. When channels did open in the presence of the peptide, they were more likely to open to substate levels. The inhibition and increased fraction of openings to subconductance levels suggested that the domain peptide might destabilise inter-domain interactions that involve the C-terminal tail. We found that Homer 1b not only interacts with the channels, but reduces the inhibitory action of the C-terminal tail peptide, perhaps by stabilizing inter-domain interactions and preventing their disruption.     

Activation of the calcium release channel (ryanodine receptor) by heparin and other polyanions is calcium dependent.

Heparin has been used as a potent competitive inhibitor of inositol 1,4,5-trisphosphate (IP3)-binding to IP3 receptors and to block IP3-gated calcium channels in bilayer experiments. In contrast to the effect on the IP3-gated channel, heparin (0.1-1 micrograms/ml) opened the Ca release channel (ryanodine receptor). Other polyanions such as pentosan polysulfate and polyvinyl sulfate also activated the Ca release channel. The effect of polyanions on the Ca release channel was Ca dependent. Polyanion addition activated the Ca release channel when free Ca was > 80 nM, but was ineffective when free Ca was < 20 nM. The level of channel activation could be altered by manipulating the free Ca concentration. These results suggest that the polyanions act by increasing the local concentration of Ca near regulatory sites on the channel complex. As most cells have both types of intracellular channels, the opposite effects of the polyanions on the two channel types suggests that addition of polyanions to intact cells may produce multiple effects.     

2-Oxoglutarate oxygenases are inhibited by a range of transition metals

2-Oxoglutarate oxygenases are inhibited by a range of transition metals, as exemplified by studies on human histone demethylases and prolyl hydroxylase domain 2 (PHD2 or EGLN1). The biological effects associated with 2-oxoglutarate oxygenase inhibition may result from inhibition of more than one enzyme and by mechanisms in addition to simple competition with the Fe(ii) cofactor.     

The metabolic effects of oxytocin are mediated by a uterine type of receptor and are inhibited by oxytocin antagonist and by arginine vasopressin in the dog.

Infusion of oxytocin (OT) into normal dogs, in doses which produced plasma levels of OT in the physiological range, has been shown to increase plasma levels of glucose, insulin and glucagon and increase rates of glucose production and uptake. This study sought to determine whether there was a correlation between these metabolic effects and the oxytocic potency of four less potent oxytocic analogues when infused into normal dogs. The rank order of oxytocic potency of all 4 correlated well with the rise in plasma glucose levels, and in 3 of the 4 with the rise in plasma insulin levels. An antagonist of the oxytocic effect of OT suppressed the usual OT-induced rise in plasma glucose, insulin and glucagon as well as the increased glucose production and uptake. Arginine vasopressin (AVP) infusion, which by itself did not produce any metabolic effects, blocked completely the effects of OT infusion to raise plasma glucose and insulin levels and increase glucose production and uptake. The data suggest that the metabolic effects of OT in the dog are mediated by OT receptors that are similar to those producing the oxytocic effects. Whether the inhibition by AVP of the metabolic and hormonal effects of OT occurs at the receptor or post receptor level or via other mechanisms remains to be determined.     

Effects of a domain peptide of the ryanodine receptor on Ca2+ release in skinned skeletal muscle fibers

Mutations in the central domain of the skeletal muscle ryanodinereceptor (RyR) cause malignant hyperthermia (MH). A synthetic peptide(DP4) in this domain (Leu-2442-Pro-2477) produces enhanced ryanodine binding and sensitized Ca²⁺ release in isolatedsarcoplasmic reticulum, similar to the properties in MH, possiblybecause the peptide disrupts the normal interdomain interactions thatstabilize the closed state of the RyR (Yamamoto T, El-Hayek R, andIkemoto N. J Biol Chem 275: 11618-11625, 2000). Here, DP4 was applied to mechanically skinned fibers of rat muscle thathad the normal excitation-contraction coupling mechanism stillfunctional to determine whether muscle fiber responsiveness wasenhanced. DP4 (100 µM) substantially potentiated the Ca²⁺release and force response to caffeine (8 mM) and to low[Mg²⁺] (0.2 mM) in every fiber examined, with nosignificant effect on the properties of the contractile apparatus. DP4also potentiated the response to submaximal depolarization of thetransverse tubular system by ionic substitution. Importantly, DP4 didnot significantly alter the size of the twitch response elicited byaction potential stimulation. These results support the proposal thatDP4 causes an MH-like aberration in RyR function and are consistentwith the voltage sensor triggering Ca²⁺ release bydestabilizing the closed state of the RyRs.

     

The effect of collagenase and temperature on mitochondrial respiratory parameters in saponin-skinned cardiac fibers

The results of a comparative study of the respiration rates of mitochondria in saponin-skinned rat cardiac fibers (SF) and in fibers treated with saponin and collagenase (SCF) suggest that only about half of the whole population of mitochondria manifest their activity in SF, in contrast to SCF, in response to extracellular substrates of oxidative phosphorylation. The apparent K_m value for ADP with succinate as substrate, which was as high as 330±32 M in SF in SF at 20 °C, decreased about 2-fold in SCF at the same temperature and in SF at 37 °C, and decreased further to 67±8 M in SCF at 37 °C. Thus, weakening or breaking of cellular contacts by collagenase and the temperature-dependence of diffusion of substrates such as ADP, seem to be important factors that determine the respiratory activity and regulatory parameters of mitochondria in saponin-permeabilized cardiomyocytes.