Intracellular generation of reactive oxygen species by contracting skeletal muscle cells

The aim of this work was to examine the intracellular generation of reactive oxygen species in skeletal muscle cells at rest and during and following a period of contractile activity. Intracellular generation of reactive oxygen species was examined directly in skeletal muscle myotubes using 2',7'-dichlorodihydrofluorescein (DCFH) as an intracellular probe. Preliminary experiments confirmed that DCFH located to the myotubes but was readily photoxidizable during repeated intracellular fluorescence measurements and strategies to minimize this were developed. The rate of oxidation of DCFH did not change significantly over 30 min in resting myotubes, but was increased by approximately 4-fold during 10 min of repetitive, electrically stimulated contractile activity. This increased rate was maintained over 10 min following the end of the contraction protocol. DCF fluorescence was distributed evenly throughout the myotube with no evidence of accumulation at any specific intracellular sites or localization to mitochondria. The rise in DCF fluorescence was effectively abolished by treatment of the myotubes with the intracellular superoxide scavenger, Tiron. Thus these data appear to represent the first direct demonstration of a rise in intracellular oxidant activity during contractile activity in skeletal muscle myotubes and indicate that superoxide, generated from intracellular sites, is the ultimate source of oxidant(s) responsible for the DCFH oxidation.     

Role of mitochondrial superoxide dismutase in contraction-induced generation of reactive oxygen species in skeletal muscle extracellular space

Contractions of skeletal muscles produce increases in concentrations of superoxide anions and activity of hydroxyl radicals in the extracellular space. The sources of these reactive oxygen species are not clear. We tested the hypothesis that, after a demanding isometric contraction protocol, the major source of superoxide and hydroxyl radical activity in the extracellular space of muscles is mitochondrial generation of superoxide anions and that, with a reduction in MnSOD activity, concentration of superoxide anions in the extracellular space is unchanged but concentration of hydroxyl radicals is decreased. For gastrocnemius muscles from adult (6–8 mo old) wild-type (Sod2^+/+) mice and knockout mice heterozygous for the MnSOD gene (Sod2^+/-), concentrations of superoxide anions and hydroxyl radical activity were measured in the extracellular space by microdialysis. A 15-min protocol of 180 isometric contractions induced a rapid, equivalent increase in reduction of cytochrome c as an index of superoxide anion concentrations in the extracellular space of Sod2^+/+ and Sod2^+/- mice, whereas hydroxyl radical activity measured by formation of 2,3-dihydroxybenzoate from salicylate increased only in the extracellular space of muscles of Sod2^+/+ mice. The lack of a difference in increase in superoxide anion concentration in the extracellular space of Sod2^+/+ and Sod2^+/- mice after the contraction protocol supported the hypothesis that superoxide anions were not directly derived from mitochondria. In contrast, the data obtained suggest that the increase in hydroxyl radical concentration in the extracellular space of muscles from wild-type mice after the contraction protocol most likely results from degradation of hydrogen peroxide generated by MnSOD activity. hydroxyl radicals; microdialysis     

Psychological impact of adjuvant chemotherapy in the first two years after mastectomy.

Psychological symptoms were assessed over two years in a randomised trial of three forms of treatment given to women after mastectomy for stage II breast cancer. The treatments were: three weeks'' radiotherapy; one year''s adjuvant chemotherapy with cyclophosphamide, methotrexate, and 5-fluorouracil; and radiotherapy followed by chemotherapy. Analysis of the results on an intention to treat basis showed no substantial differences in depression or anxiety among groups at one, three, or six months after the operation. At 13 months, however, patients who had been allocated chemotherapy had significantly more symptoms, especially depression, than control patients treated with radiotherapy alone. Conditioned reflex nausea and vomiting increased considerably during the second six months of chemotherapy and persisted for up to a year afterwards. The psychological morbidity of adjuvant chemotherapy could be substantially reduced if courses of treatment were restricted to about six months.     

Uptake of copper by mouse hepatocytes

This study has investigated the uptake of copper by mouse hepatocytes. The cells gave similar results whether they were used right after isolation or maintained overnight on collagen-coated dishes. Uptake from cells in suspension followed two phases: an initial rapid binding followed by a linear uptake phase. The two phases were not so easily distinguishable in cells grown in culture where uptake was linear over the first hour. The uptake showed saturation but may not have followed simple kinetics. Histidine stimulated uptake in a concentration-dependent manner, as did some other amino acids, but copper had very little effect on histidine uptake. The process was not dependent on intracellular adenosine triphosphate (ATP), since inhibitors that substantially reduced ATP levels inside the cell did not alter copper uptake. The inhibitors, however, blocked histidine uptake to varying degrees, suggesting that copper and histidine are taken up by different pathways. The uptake was reduced markedly by N-ethyl maleimide, and preincubation of the cells with "Pronase" resulted in a decrease of uptake. A model for the uptake of copper by hepatocytes that incorporates the data presented in this paper with that produced by earlier workers is suggested.     

The Human Gonadotropin Releasing Hormone Type I Receptor Is a Functional Intracellular GPCR Expressed on the Nuclear Membrane

The mammalian type I gonadotropin releasing hormone receptor (GnRH-R) is a structurally unique G protein-coupled receptor (GPCR) that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME). Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER) leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS) in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor.     

Time course of responses of human skeletal muscle to oxidative stress induced by nondamaging exercise.

Previous studies in animals have demonstrated that a single period of aerobic exercise induces a rise in the skeletal muscle activity of the antioxidant enzymes superoxide dismutase and catalase and an increase in the muscle content of heat shock proteins (HSPs). The purpose of this study was to examine the time course of response of human skeletal muscle superoxide dismutase and catalase activities and the content of HSP60 and HSP70 after a period of exhaustive, nondamaging aerobic exercise. Seven volunteers undertook one-legged cycle ergometry at 70% maximal oxygen uptake for 45 min. Biopsies were obtained from the vastus lateralis muscle 7 days before and at 1, 2, 3, and 6 days after exercise. Muscle superoxide dismutase activity increased to a peak at 3 days postexercise, muscle catalase activities were unchanged, and muscle content of HSP60 and the inducible HSP70 increased by variable amounts to reach means of 190% and 3,100% of preexercise values, respectively, by 6 days postexercise. These data indicate that human skeletal muscle responds to a single bout of nondamaging exercise by increasing superoxide dismutase activity and provide the first evidence of an increase in HSP content of human skeletal muscle after a submaximal exercise bout.     

Signaling to Extracellular Signal-regulated Kinase from ErbB1 Kinase and Protein Kinase C: FEEDBACK,HETEROGENEITY, AND GATING*

Many extracellular signals act via the Raf/MEK/ERK cascade in which kinetics, cell-cell variability, and sensitivity of the ERK response can all influence cell fate. Here we used automated microscopy to explore the effects of ERK-mediated negative feedback on these attributes in cells expressing endogenous ERK or ERK2-GFP reporters. We studied acute rather than chronic stimulation with either epidermal growth factor (ErbB1 activation) or phorbol 12,13-dibutyrate (PKC activation). In unstimulated cells, ERK-mediated negative feedback reduced the population-average and cell-cell variability of the level of activated ppERK and increased its robustness to changes in ERK expression. In stimulated cells, negative feedback (evident between 5 min and 4 h) also reduced average levels and variability of phosphorylated ERK (ppERK) without altering the “gradedness” or sensitivity of the response. Binning cells according to total ERK expression revealed, strikingly, that maximal ppERK responses initially occur at submaximal ERK levels and that this non-monotonic relationship changes to an increasing, monotonic one within 15 min. These phenomena occur in HeLa cells and MCF7 breast cancer cells and in the presence and absence of ERK-mediated negative feedback. They were best modeled assuming distributive (rather than processive) activation. Thus, we have uncovered a novel, time-dependent change in the relationship between total ERK and ppERK levels that persists without negative feedback. This change makes acute response kinetics dependent on ERK level and provides a “gating” or control mechanism in which the interplay between stimulus duration and the distribution of ERK expression across cells could modulate the proportion of cells that respond to stimulation.     

Isolation and properties of sarcoplasmic reticulum from the fast muscles of plaice Pleuronectes platessa L.

This paper describes a method for the isolation of highly purified sarcoplasmic reticulum from plaice fast muscle. The interrelationships of pH, KCL, Ca²⁺, Mg²⁺, ADP and temperature have been investigated. Protein composition of plaice white muscle sarcoplasmic reticulum was found to be comparable to that described for rabbit fast muscle, with a major component of 100 000 daltons. Arrhenius plots of the Ca²⁺-AT Pase are linear over the range 0–30°C. Activation enthalpy (60±1.5 kJ/mol) was found to be independent of KCl concentration. The calcium concentration required to give half maximal activation of the AT Pase (K_Ca) was found to decrease with increasing temperature, from a maximum of 1.7 μm at 0°C to 0.55 at 20°C.     

Beta-adrenergic-receptor-mediated suppression of interleukin 2 receptors in human lymphocytes

Adrenergic receptor agonists are known to attenuate the proliferative response of human lymphocytes after activation; however, their mechanism of action is unknown. Since expression of interleukin 2 (IL-2) receptors is a prerequisite for proliferation, the effect of beta-adrenergic receptor agonists on lymphocyte IL-2 receptors was studied on both mitogen-stimulated lymphocytes and IL-2-dependent T lymphocyte cell lines. In both cell types the beta-adrenergic receptor agonist isoproterenol blocked the expression of IL-2 receptors, as determined with the IL-2 receptor anti-TAC antibody. To determine the effect of beta-adrenergic agonists on expression of the high affinity IL-2 receptors, [125I]IL-2 binding studies were performed at concentrations selective for high affinity sites. No significant effect of beta-adrenergic agonists on high affinity IL-2 receptor sites could be detected. The data demonstrate that beta-adrenergic receptor agonists down-regulate IL-2 receptors primarily affecting low affinity sites.