The toxic effects of anticholinesterases on muscle

It has been shown that acute administration of ecothiopate iodine in vivo caused an approximate 80% depression of acetylcholinesterase activity in the diaphragms of mice. Inhibition of acetylcholinesterase was accompanied by an influx of calcium at the junctional region of the diaphragm, which continued during subsequent progressive development of a severe myopathy located in the same region. Myopathy was accompanied by loss of creatine kinase from the muscle and was represented, at the light microscope level, by hypercontraction, Procion Yellow staining and loss of cross striations within the muscle fibres. It appeared to reach a point of maximum severity approximately 3-6 hours after ecothiopate administration and then, by means of some repair/regeneration process, regained an apparently normal morphology within 72 hours of the intoxication. At the ultrastructural level, ecothiopate-induced myopathy was recognised by loss of Z-lines, swelling and vacuolation of mitochondria and sarcoplasmic reticulum, dissarray of myofilaments, crystal formation, and sometimes, by the complete obliteration of sarcomeric structure. The development of myopathy in vitro was shown to be nerve-mediated and to require a functional acetylcholine receptor for its development It was successfully treated therapeutically in vivo by pyridine-2-aldoxime methiodide and prophylactically by pyridostigmine bromide. However, the use of a range of membrane-on channel blockers, and of leupeptin, an inhibitor of calcium-activated-neutral-protease, have been unsuccessful in the prevention of ecothiopate-induced myopathy.

Modulation of SERCA in the chronic phase of adjuvant arthritis as a possible adaptation mechanism of redox imbalance

Adjuvant arthritis (AA) is a condition that involves systemic oxidative stress. Unexpectedly, it was found that sarcoplasmic reticulum Ca2 +-ATPase (SERCA) activity was elevated in muscles of rats with AA compared to controls, suggesting possible conformational changes in the enzyme. There was no alteration in the nucleotide binding site but rather in the transmembrane domain according to the tryptophan polar/non-polar fluorescence ratio. Higher relative expression of SERCA, higher content of nitrotyrosine but no increase in phospholipid oxidation in AA SR was found. In vitro treatments of SR with HOCl showed that in AA animals SERCA activity was more susceptible to oxidative stress, but SR phospholipids were more resistant and SERCA could also be activated by phosphatidic acid. It was concluded that increased SERCA activity in AA was due to increased levels of SERCA protein and structural changes to the protein, probably induced by direct and specific oxidation involving reactive nitrogen species.

Mechanism of serca modulation from rats with adjuvant arthritis

We studied the structural and functional alterations of SERCA in rats suffering from adjuvant arthritis (AA). AA was induced by intradermal administration of Mycobacterium butyricum (MB) to the base of the tail of Lewis rats. Injury of SERCA from skeletal muscles of AA rats was analyzed on days 7, 14, 21 and 28 after MB injection. Neither fragmentation, aggregation of SERCA protein, alterations in SH groups, nor oxidation of phosphatidylcholines and phosphatidylethanolamines in SR vesicles were observed in animals with AA. The only ROS/RNS modification was increased formation of nitrotyrosine. The activity of SERCA from AA animals decreased on day 21 after MB injection and was associated with a significant increase of protein carbonyls in sarcoplasmic reticulum (SR). In contrast, on day 28 an increase of SERCA activity was observed and protein carbonyl level reversed to control level. Concerning kinetic parameters, maximum reaction velocity (Vmax) decrease and increase was observed with respect to both substrates (Ca, ATP) on days 21 and 28, respectively, suggesting possible conformational changes of the enzyme. These changes were not associated with alterations in nucleotide binding site situated in cytosol, but rather with tryptophan fluorescence intensity ratio (cytosol/membrane) related to the transmembrane domain of SERCA. Elevated SERCA activity on day 28 was caused by its higher expression. Acidic phospholipids (PA), probably present in SR of AA rats, may contribute to the elevation of Ca-ATPase activity, as PA administration in vitro increased this activity.

Detection and quantification of protein oxidation in sarcopenic models:a mass spectrometry study

Oxidised biomolecules in aged tissue could potentially be used as biomarkers for age-related diseases; however, it is still unclear whether they causatively contribute to ageing or are consequences of the ageing process. To assess the potential of using protein oxidation as markers of ageing, mass spectrometry (MS) was employed for the identification and quantification of oxidative modifications in obese (ob/ob) mice. Lean muscle mass and strength is reduced in obesity, representing a sarcopenic model in which the levels of oxidation can be evaluated for different muscular systems including calcium homeostasis, metabolism and contractility. Several oxidised residues were identified by tandem MS (MS/MS) in both muscle homogenate and isolated sarcoplasmic reticulum (SR), an organelle that regulates intracellular calcium levels in muscle. These modifications include oxidation of methionine, cysteine, tyrosine, and tryptophan in several proteins such as sarcoplasmic reticulum calcium ATPase (SERCA), glycogen phosphorylase, and myosin. Once modifications had been identified, multiple reaction monitoring MS (MRM) was used to quantify the percentage modification of oxidised residues within the samples. Preliminary data suggests proteins in ob/ob mice are more oxidised than the controls. For example SERCA, which constitutes 60-70% of the SR, had approximately a 2-fold increase in cysteine trioxidation of Cys561 in the obese model when compared to the control. Other obese muscle proteins have also shown a similar increase in oxidation for various residues. Further analysis with complex protein mixtures will determine the potential diagnostic use of MRM experiments for analysing protein oxidation in small biological samples such as muscle needle biopsies.

Endothelial cell redox regulation of ischemic angiogenesis

The endothelium produces and responds to reactive oxygen and nitrogen species (RONS), providing important redox regulation to the cardiovascular system in physiology and disease. In no other situation are RONS more critical than in the response to tissue ischemia. Here, tissue healing requires growth factor-mediated angiogenesis that is in part dependent on low levels of RONS, which paradoxically must overcome the damaging effects of high levels of RONS generated as a result of ischemia. While generation of endothelial cell RONS in hypoxia/reoxygenation is acknowledged, the mechanism for their role in angiogenesis is still poorly understood. During ischemia, the major low molecular weight thiol glutathione (GSH) reacts with RONS and protein cysteines, producing GSH-protein adducts. Recent data indicate that GSH adducts on certain proteins are essential to growth factor responses in endothelial cells. Genetic deletion of the enzyme glutaredoxin-1, which selectively removes GSH protein adducts, improves, while its overexpression impairs, revascularization of the ischemic hindlimb of mice. Ischemia-induced GSH adducts on specific cysteine residues of several proteins, including p65 NFkB and the sarcoplasmic reticulum calcium ATPase-2 (SERCA2), appear to promote ischemic angiogenesis. Identifying the specific proteins in the redox response to ischemia has provided therapeutic opportunities to improve clinical outcomes of ischemia.

Temperature-related differences in the calcium transient between Atlantic cod (Gadus morhua) and steelhead trout (Oncorhynchus mykiss) cardiomyocytes

The Atlantic cod, Gadus morhua, differs from many teleosts in that its heart does not respond to adrenergic stimulation, and is more capable of maintaining function during acute temperature changes. To examine if differences in intracellular calcium mobilization are associated with these atypical responses, confocal microscopy was used to study the calcium handling of cardiac cells from Atlantic cod vs. steelhead trout at their acclimation temperature (10ºC), or subjected to acute temperature changes (to 4 and 16ºC), while being stimulated across a range of frequencies (10 – 110 min⁻¹). In addition, cells were tested with and without tonic (10 nM) levels of adrenaline at 10ºC, and pharmacological blockers were used to study the relative contributions of the L-type Ca²⁺ channel, sarcoplasmic reticulum and Na+/Ca²⁺ exchanger to the Ca²⁺ transient. Consistent with previous in vitro and in situ studies, there were few significant effects of adrenaline on the Ca²⁺ transient of cod cardiomyocytes, yet adrenaline had significant positive inotropic effects on trout cardiomyocytes. At 10ºC, peak Ca²⁺ (F/F₀) only differed between the two species at low stimulation frequencies (10, 30 min-1), with trout F/F₀ 25-35% higher. In contrast, the time to peak Ca²⁺ and the time to half relaxation were both shorter (by 10 – 35% across frequencies) in cod. Acute temperature changes caused a shift in the Ca²⁺ - frequency relationship in both species, with F/F₀ values higher for trout at low frequencies (< 70 min⁻¹) at 4ºC, whereas this parameter was greater at all frequencies except 10 min⁻¹ in cod at 16ºC. Unfortunately, these experiments did not highlight clear species differences in the relative contributions of the L-type Ca²⁺ channels, sarcoplasmic reticulum and Na+/Ca²⁺ exchange to the Ca²⁺ transient.

Spongionella Secondary Metabolites Regulate Store Operated Calcium Entry Modulating Mitochondrial Functioning in SH-SY5Y Neuroblastoma Cells

cknowledgements The research leading to these results has received funding from the following FEDER cofounded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and Consellería de Cultura, Educación e OrdenaciónUniversitaria, GRC2013-016, and through AxenciaGalega de Innovación, Spain, ITC-20133020 SINTOX. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union's Seventh Framework Programme managed by REA - Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEA. Jon Andoni Sánchez is supported by a fellowship from Plan Galego de Investigación e Crecemento, Xunta de Galicia, Spain.

Type 1 ryanodine receptor interactions

Excitation-contraction coupling is an essential part of skeletal muscle contraction. It encompasses the sensing of depolarisation of the plasma membrane coupled with the release of Ca2+ from intracellular stores. The channel responsible for this release is called the Ryanodine receptor (RyR), and forms a hub of interacting proteins which work in concert to regulate the release of Ca2+ through this channel. The aim of this work was to characterise possible novel interactions with a proline-rich region of the RyR1, to characterise a monoclonal antibody (mAb VF1c) raised against a junctional sarcoplasmic reticulum protein postulated to interact with RyR1, and to characterise the protein recognised by this antibody in models of skeletal muscle disease such as Duchenne Muscular dystrophy (DMD) and sarcopenia. These experiments were performed using cell culture, protein purification via immunoprecipitation, affinity purification, low pressure chromatography and western blotting techniques. It was found that the RyR1 complex isolated from rat skeletal muscle co-purifies with the Growth factor receptor bound protein 2 (GRB2), very possibly via an interaction between the proline rich region of RyR1 and one of the SH3 domains located on the GRB2 protein. It was also found that Pleiotrophin and Phospholipase Cγ1, suggested interactors of the proline rich region of RyR1, did not co-purify with the RyR1 complex. Characterisation of mAb VF1c determined that this monoclonal antibody interacts with junctophilin 1, and binds to this protein between the region of 369-460, as determined by western blotting of JPH1 fragments expressed in yeast. It was also found that JPH1 and JPH2 are differentially regulated in different muscles of rabbit, where the highest amount of both proteins was found in the extensor digitorum longus (EDL) muscle. JPH1 and 2 levels were also examined in three rodent models of disease: the mdx mouse (a model of DMD), chronic intermittent hypoxia (CIH)-treated rat, and aged and adult mice, a model of sarcopenia. In the EDL and soleus muscle of CIH treated rats, no difference in either JPH1 or JPH2 abundance was detected in either muscle. An examination of JPH1 and 2 expression in mdx and wild type controls diaphragm, vastus lateralis, soleus and gastrocnemius muscle found no major differences in JPH1 abundance, while JPH2 was decreased in mdx gastrocnemius compared to wild type. In a mouse model of sarcopenia, JPH1 abundance was found to be increased in aged soleus but not in aged quadriceps, while in exercised quadriceps, JPH2 abundance was decreased compared to unexercised controls. Taken together, these results have implications for the regulation of RyR1 and JPH1 and 2 in skeletal muscle in both physiological and pathological states, and provide a newly characterised antibody to expand the field of JPH1 research.

Distribution of diatom species as abundance estimates from vertical multinet hauls from 150 m water depth in the Indian Ocean

During the Indian Ocean Expedition of R/V METEOR phytoplankton samples were taken with a multiple closing net (Multinet) at 103 stations. In this material the diatoms were investigated. In all 247 taxa could be identified which belong to 242 species and 5 varieties of formae of 80 genera. Of these 1 variety, 15 pecies, and 3 genera are newly described. New combinations were made for 18 species, and a number of old combinations was reinstated.

Effects of menthol on rat tail artery mediated by TRPM8 and voltage-gated calcium channels

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold sensing role in sensory neurons, it is expressed and functional in several non-neuronal tissues, including vasculature. We previously demonstrated that menthol causes vasoconstriction and vasodilatation in isolated arteries, depending on vascular tone. Here we investigated calcium's role in responses mediated by TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology and ratiometric Ca2+ recording. Isometric contraction studies examined actions of TRPM8 ligands in the presence/absence of L-type calcium channel blocker. Menthol (300 μM), a concentration typically used to induce TRPM8 currents, strongly inhibited L-type voltage-dependent Ca2+ current (L-ICa) in myocytes, especially it's sustained component, most relevant for depolarisation-induced vasoconstriction. In contraction studies, with nifedipine present (10 μM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol. Menthol-induced increases in PE-induced vasoconstrictions were mediated predominantly by Ca2+-release from sarcoplasmic reticulum, since they were significantly inhibited by cyclopiazonic acid. Pre-incubation of vascular rings with a TRPM8 antagonist strongly inhibited menthol-induced increases in PE-induced vasoconstrictions, thus confirming specific role of TRPM8. Finally, two other common TRPM8 agonists, WS-12 and icilin, inhibited L-ICa. Thus, TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels, and largely obscure TRPM8-mediated vasoconstriction.

Identification of Genes Modulating Mitochondrial Biogenesis in Caenorhabditis elegans

Since Altmann recognized ubiquitously distributed "bioblasts" in 1890, understanding of mitochondria has evolved from "elementary organisms" living inside cells and carrying out vital functions, over the Harman's "free radical theory" in 1956, to one of the driving forces of aging and cause of multiple associated diseases impacting society today. While a tremendous amount of work has contributed to the understanding of mitochondrial biology in different model organisms, the precise molecular mechanisms of basic mitochondrial function have yet to be deciphered. By employing an RNA interference mediated screen in Caenorhabditis elegans, we identified two transcription factors: SPTF-3, a member of Sp1 family, and an uncharacterized, nematode specific W04D2.4. We propose that both proteins modulate expression of many genes with regard to mitochondrial function including mitochondrial single-stranded binding protein encoded by mtss-1, whose promoter was used as transcriptional reporter in the screen. Further, RNA sequencing data indicate that W04D2.4 indirectly regulates expression of mitochondrial DNA via control of genes functionally related to mitochondrial replication and translation machineries. We also demonstrate that from all interventions targeting cytosolic translation, MTSS-1 levels are elevated only upon knockdown of genes encoding cytosolic ribosomal proteins. Reduction of ribosomes leads to increased sptf-3 translation, most likely in an internal ribosome entry side (IRES) mediated manner, eventually inducing mtss-1 expression. Moreover, we identify a novel role for SPTF-3 in the regulation of mitochondrial unfolded stress response (UPRmt) activation, but not endoplasmatic reticulum or oxidative stress responses. Taken together, this study identifies two transcription factors previously not associated with mitochondrial biogenesis and UPRmt in C. elegans, establishing a basis for further investigation of mito-nuclear interactions.

C-terminal residues of skeletal muscle calsequestrin are essential for calcium binding and for skeletal ryanodine receptor inhibition

Calcium response of KCl-excited populations of ventricular myocytes from the European sea bass (Dicentrarchus labrax): a promising approach to integrate cell-to-cell heterogeneity in studying the cellular basis of fish cardiac performance

Climate change challenges the capacity of fishes to thrive in their habitat. However, through phenotypic diversity, they demonstrate remarkable resilience to deteriorating conditions. In fish populations, inter-individual variation in a number of fitness-determining physiological traits, including cardiac performance, is classically observed. Information about the cellular bases of inter-individual variability in cardiac performance is scarce including the possible contribution of excitation-contraction (EC) coupling. This study aimed at providing insight into EC coupling-related Ca2+ response and thermal plasticity in the European sea bass (Dicentrarchus labrax). A cell population approach was used to lay the methodological basis for identifying the cellular determinants of cardiac performance. Fish were acclimated at 12 and 22 A degrees C and changes in intracellular calcium concentration ([Ca2+](i)) following KCl stimulation were measured using Fura-2, at 12 or 22 A degrees C-test. The increase in [Ca2+](i) resulted primarily from extracellular Ca2+ entry but sarcoplasmic reticulum stores were also shown to be involved. As previously reported in sea bass, a modest effect of adrenaline was observed. Moreover, although the response appeared relatively insensitive to an acute temperature change, a difference in Ca2+ response was observed between 12- and 22 A degrees C-acclimated fish. In particular, a greater increase in [Ca2+](i) at a high level of adrenaline was observed in 22 A degrees C-acclimated fish that may be related to an improved efficiency of adrenaline under these conditions. In conclusion, this method allows a rapid screening of cellular characteristics. It represents a promising tool to identify the cellular determinants of inter-individual variability in fishes' capacity for environmental adaptation.

Interacção de complexos de vanádio-citrato com a bomba de cálcio de retículo sarcoplasmático

Dissertação de mest. em Biotecnologia, Departamento de Química e Bioquímica da Faculdade de Ciêicias e Tecnologia, Univ. do Algarve, 2004