Intracellular signalling pathways regulating the adaptation of skeletal muscle to exercise and nutritional changes

The focus of the present review is to assimilate current knowledge concerning the differing signalling transduction cascades that control muscle mass development and affect skeletal muscle phenotype following exercise or nutritional uptake. Effects of mechanical loading on protein synthesis are discussed. Muscle growth control is regulated by the interplay of growth promoting and growth suppressing factors, which act in concert. Much emphasis has been placed on understanding how increases in the rate of protein synthesis are induced in skeletal muscle during the adaptive process. One key point to emerge is that protein synthesis following resistance exercise or increased nutrient availability is mediated through changes in signal transduction involving the phosphorylation of mTOR and sequential activation of downstream targets. On the other hand, AMPK activation plays an important role in the inhibition of protein synthesis by suppressing the function of multiple translation regulators of the mTOR signalling pathway in response to cellular energy depletion and low metabolic conditions. The effects of exercise and/or nutritional uptake on the activation of signalling molecules that regulate protein synthesis are highlighted, providing a better understanding of the molecular changes in the cell.

Expression and regulation of Nkd-1, an intracellular component of Wnt signalling pathway in the chick embryo

The Wnt family of secreted signalling molecules control a wide range of developmental processes in all metazoans. The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement. Recent work has identified the protein Naked Cuticle to act as an intracellular switch to promote the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked Cuticle-1 (cNkd-1) and show that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly, we show that the expression of cNkd-1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

Modulation of peroxynitrite-induced fibroblast injury by hesperetin: a role for intracellular scavenging and modulation of ERK signalling

Peroxynitrite is thought to contribute to the progression of many diseases including cardiovascular disease, cancer, and neurodegenerative disorders. We report that pre-treatment of fibroblasts with the citrus flavanone, hesperetin, prior to peroxynitrite exposure protects against peroxynitrite-mediated cytotoxicity. This protection was partially mediated by the intracellular scavenging of peroxynitrite by hesperctin as exposure of fibroblasts to peroxynitrite following hesperetin loading led to the formation of two intracellular nitrohesperetin derivatives. In addition, protection appeared to be mediated by hesperetin-induced changes in MAP kinase signalling. Exposure of fibroblasts to hesperetin led to concentration-dependent increases in the phosphorylation of ERK1/2 and was observed to restore peroxynitrite-mediated decreases in ERK1/2 phosphorylation. We propose that the protective potential of hesperetin in fibroblasts may be mediated both by intracellular scavenging of peroxynitrite and by modulation of fibroblast signalling. (c) 2006 Elsevier Inc. All rights reserved.

The intracellular genistein metabolite 5,7,3 ',4 '-tetrahydroxyisoflavone mediates G2-M cell cycle arrest in cancer cells via modulation of the p38 signaling pathway

The cellular actions of genistein are believed to mediate the decreased risk of breast cancer associated with high soy consumption. We have investigated the intracellular metabolism of genistein in T47D tumorigenic and MCF-10A nontumorigenic cells and assessed the cellular actions of resultant metabolites. Genistein selectively induced growth arrest and G2-M phase cell cycle block in T47D but not MCF10A breast epithelial cells. These antiproliferative effects were paralleled by significant differences in the association of genistein to cells and in particular its intracellular metabolism. Genistein was selectively taken up into T47D cells and was subject to metabolism by CYP450 enzymes leading to the formation of both 5,7,3',4'-tetrahydroxyisoflavone (THIF) and two glutathionyl conjugates of THIF THIF inhibited cdc2 activation via the phosphorylation of p38 MAP kinase, suggesting that this species may mediate genistein's cellular actions. THIF exposure activated p38 and caused subsequent inhibition of cyclin B1 (Ser 147) and cdc2 (Thr 161) phosphorylation, two events critical for the correct functioning of the cdc2-cyclin B1 complex. We suggest that the formation of THIF may mediate the cellular actions of genistein in tumorigenic breast epithelial cells via the activation of signaling through p38. (c) 2006 Elsevier Inc. All rights reserved.

Intracellular metabolism and bioactivity of quercetin and its in vivo metabolites

Understanding the cellular effects of flavonoid metabolites is important for predicting which dietary flavonoids might be most beneficial in vivo. Here we investigate the bioactivity in dermal fibroblasts of the major reported in vivo metabolites of quercetin, i.e. 3'-O-methyl quercetin, 4'-O-methyl quercetin and quercetin 7-O-beta-D-glucuronide, relative to that of quercetin, in terms of their further metabolism and their resulting cytotoxic and/or cytoprotective effects in the absence and presence of oxidative stress. Uptake experiments indicate that exposure to quercetin led to the generation of two novel cellular metabolites, one characterized as a 2'-glutathionyl quercetin conjugate and another product with similar spectral characteristics but 1 mass unit lower, putatively a quinone/quinone methide. A similar product was identified in cells exposed to 3'-O-methyl quercetin, but not in the lysates of those exposed to its 4'-O-methyl counterpart, suggesting that its formation is related to oxidative metabolism. There was no uptake or metabolism of quercetin 7-O-beta-D-glucuronide by fibroblasts. Formation of oxidative metabolites may explain the observed concentration-dependent toxicity of quercetin and 3'-O-methyl quercetin, whereas the formation of a 2'-glutathionyl quercetin conjugate is interpreted as a detoxification step. Both O -methylated metabolites conferred less protection than quercetin against peroxide-induced damage, and quercetin glucuronide was ineffective. The ability to modulate cellular toxicity paralleled the ability of the compounds to decrease the level of peroxide-induced caspase-3 activation. Our data suggest that the actions of quercetin and its metabolites in vivo are mediated by intracellular metabolites.

Intracellular or intercellular localization of the polar pathway of penetration across stratum corneum

A pharmacokinetic hypothesis of stratum corneum with two parallel pathways, lipophilic and porous hydrophilic, is not well documented yet. Still questionable is the localization of the pores, and the present experiments were designed to elucidate the contribution of extracellular lipids and intracellular keratin to the structure of this pathway. Percutaneous penetration of baclofen, a model zwitterion, was studied in vitro using human cadaver skin. Aqueous or ethanolic saturated solutions of the drug (Cs = 4.6 and 0.4 mg/ mL, respectively) were applied on the skin that was pretreated with: methanol/chloroform (Me/Ch) or acetone-chloroform (Ac/Ch) (1:1) mixtures, or with these solvents followed by 0.2% solution of sodium lauryl sulfate (SLS). As controls, baclofen penetration through the intact full-thickness skin was determined, and the fluxes were 0.18 ±0.08 and 0.14 ±0.07 µg/cm2/h for aqueous and ethanolic solutions, respectively. When Me/Ch was used for 1 h, an expected increase of the penetration was observed, but the lag time, Tlag, was still nearly 20 h. When the less polar mixture, Ac/Ch, was used, no flux enhancement was observed, and with ethanol as the vehicle, decreased penetration was even noted. No effect on baclofen penetration was observed when SLS was used for 1 h after delipidization of the skin was done with either the Me/Ch or Ac/Ch mixture. The results suggest that the polar pathway may be located intercellularly and comprises aqueous regions surrounded by polar lipids, which create the walls of such microchannels.

The SV2A ligand levetiracetam inhibits presynaptic Ca2+ channels via an intracellular pathway

Levetiracetam (LEV) is a prominent antiepileptic drug (AED) which binds to neuronal synaptic vesicle glycoprotein 2A (SV2A) protein and has reported effects on ion channels, but retains a poorly-defined mechanism of action. Here, we investigate inhibition of voltage-dependent Ca2+ (CaV) channels as a potential mechanism by which LEV imparts effects on neuronal activity. We used electrophysiological methods to investigate the effects of LEV on cholinergic synaptic transmission and CaV channel activity in superior cervical ganglion neurons (SCGNs). In parallel, we investigated effects of the LEV ‘inactive’ R-enantiomer, UCB L060. Thus, LEV, but not UCB L060 (each 100 μM), inhibited synaptic transmission between SCGNs in long-term culture in a time-dependent manner, significantly reducing excitatory postsynaptic potentials (EPSP) following ≥30 min application. In isolated SCGNs, LEV pretreatment (≥1 h), but not acute (5 min) application, significantly inhibited whole-cell IBa amplitude. In current clamp recordings, LEV reduced the amplitude of the afterhyperpolarizing potential (AHP) in a Ca2+-dependent manner, but also increased action potential (AP) latency in a Ca2+-independent manner, suggesting further mechanisms associated with reduced excitability. Intracellular LEV application (4-5 min) caused a rapid inhibition of IBa amplitude to an extent comparable to that seen following extracellular LEV pretreatment ( ≥ 1 h). Neither pretreatment nor intracellular application of UCB L060 produced any inhibitory effects on IBa amplitude. These results identify a stereospecific intracellular pathway by which LEV inhibits presynaptic CaV channels; resultant reductions in neuronal excitability are proposed to contribute to the anticonvulsant effects of LEV.

Intracellular accumulation of high levels of gamma-aminobutyrate by Listeria monocytogenes 10403S in response to low pH: uncoupling of gamma-aminobutyrate synthesis from efflux in a chemically defined medium

It is well established that the glutamate decarboxylase (GAD) system is central to the survival of Listeria monocytogenes at low pH, both in acidic foods and within the mammalian stomach. The accepted model proposes that under acidic conditions extracellular glutamate is transported into the cell in exchange for an intracellular gamma-aminobutyrate (GABA(i)). The glutamate is then decarboxylated to GABA(i), a reaction that consumes a proton, thereby helping to prevent acidification of the cytoplasm. In this study, we show that glutamate supplementation had no influence on either growth rate at pH 5.0 or survival at pH 2.5 when L. monocytogenes 10403S was grown in a chemically defined medium (DM). In response to acidification, cells grown in DM failed to efflux GABA, even when glutamate was added to the medium. In contrast, in brain heart infusion (BHI), the same strain produced significant extracellular GABA (GABA(e)) in response to acidification. In addition, high levels of GABA(i) (>80 mM) were found in the cytoplasm in response to low pH in both growth media. Medium-swap and medium-mixing experiments revealed that the GABA efflux apparatus was nonfunctional in DM, even when glutamate was present. It was also found that the GadT2D2 antiporter/decarboxylase system was transcribed poorly in DM-grown cultures while overexpression of gadD1T1 and gadD3 occurred in response to pH 3.5. Interestingly, BHI-grown cells did not respond with upregulation of any of the GAD system genes when challenged at pH 3.5. The accumulation of GABA(i) in cells grown in DM in the absence of extracellular glutamate indicates that intracellular glutamate is the source of the GABA(i). These results demonstrate that GABA production can be uncoupled from GABA efflux, a finding that alters the way we should view the operation of bacterial GAD systems.

Characterisation of the intracellular-glutamate decarboxylase system (GAD): analysis of its function, transcription and role in the acid resistance of various strains of Listeria monocytogenes

The glutamate decarboxylase (GAD) system is important for the acid resistance of Listeria monocytogenes. We previously showed that under acidic conditions, glutamate (Glt)/γ-aminobutyrate (GABA) antiport is impaired in minimal media but not in rich ones, like brain heart infusion. Here we demonstrate that this behavior is more complex and it is subject to strain and medium variation. Despite the impaired Glt/GABA antiport, cells accumulate intracellular GABA (GABA(i)) as a standard response against acid in any medium, and this occurs in all strains tested. Since these systems can occur independently of one another, we refer to them as the extracellular (GAD(e)) and intracellular (GAD(i)) systems. We show here that GAD(i) contributes to acid resistance since in a ΔgadD1D2 mutant, reduced GABA(i) accumulation coincided with a 3.2-log-unit reduction in survival at pH 3.0 compared to that of wild-type strain LO28. Among 20 different strains, the GAD(i) system was found to remove 23.11% ± 18.87% of the protons removed by the overall GAD system. Furthermore, the GAD(i) system is activated at milder pH values (4.5 to 5.0) than the GAD(e) system (pH 4.0 to 4.5), suggesting that GAD(i) is the more responsive of the two and the first line of defense against acid. Through functional genomics, we found a major role for GadD2 in the function of GAD(i), while that of GadD1 was minor. Furthermore, the transcription of the gad genes in three common reference strains (10403S, LO28, and EGD-e) during an acid challenge correlated well with their relative acid sensitivity. No transcriptional upregulation of the gadT2D2 operon, which is the most important component of the GAD system, was observed, while gadD3 transcription was the highest among all gad genes in all strains. In this study, we present a revised model for the function of the GAD system and highlight the important role of GAD(i) in the acid resistance of L. monocytogenes.

A modified rapid enzymatic microtiter plate assay for the quantification of intracellular γ-aminobutyric acid and succinate semialdehyde in bacterial cells

The GABase assay is widely used to rapidly and accurately quantify levels of extracellular γ-aminobutyric acid (GABA). Here we demonstrate a modification of this assay that enables quantification of intracellular GABA in bacterial cells. Cells are lysed by boiling and ethanolamine-O-sulphate, a GABA transaminase inhibitor is used to distinguish between GABA and succinate semialdehyde.

A multi-level analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Diaminofluoresceins are widely used probes for detection and intracellular localization of NO formation in cultured/isolated cells and intact tissues. The fluorinated derivative, 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), has gained increasing popularity in recent years due to its improved NO-sensitivity, pH-stability, and resistance to photo-bleaching compared to the first-generation compound, DAF-2. Detection of NO production by either reagent relies on conversion of the parent compound into a fluorescent triazole, DAF-FM-T and DAF-2-T, respectively. While this reaction is specific for NO and/or reactive nitrosating species, it is also affected by the presence of oxidants/antioxidants. Moreover, the reaction with other molecules can lead to the formation of fluorescent products other than the expected triazole. Thus additional controls and structural confirmation of the reaction products are essential. Using human red blood cells as an exemplary cellular system we here describe robust protocols for the analysis of intracellular DAF-FM-T formation using an array of fluorescence-based methods (laser-scanning fluorescence microscopy, flow cytometry and fluorimetry) and analytical separation techniques (reversed-phase HPLC and LC-MS/MS). When used in combination, these assays afford unequivocal identification of the fluorescent signal as being derived from NO and are applicable to most other cellular systems without or with only minor modifications.

Oxidative stress and growth-regulating intracellular signaling pathways in cardiac myocytes

The toxic effects of oxidative stress on cells (including cardiac myocytes, the contractile cells of the heart) are well known. However, an increasing body of evidence has suggested that increased production of reactive oxygen species (ROS) promotes cardiac myocyte growth. Thus, ROS may be 'second messenger' molecules in their own right, and growth-promoting neurohumoral agonists might exert their effects by stimulating production of ROS. The authors review the principal growth-promoting intracellular signaling pathways that are activated by ROS in cardiac myocytes, namely the mitogen-activated protein kinase cascades (extracellular signal-regulated kinases 1/2, c-Jun N-terminal kinases, and p38-mitogen-activated protein kinases) and the phosphoinositide 3-kinase/protein kinase B (Akt) pathway. Possible mechanisms are discussed by which these pathways are activated by ROS, including the oxidation of active site cysteinyl residues of protein and lipid phosphatases with their consequent inactivation, the potential involvement of protein kinase C or the apoptosis signal-regulating kinase 1, and the current models for the activation of the guanine nucleotide binding protein Ras.

Oleaginous yeast Cryptococcus curvatus exhibits interplay between biosynthesis of intracellular sugars and lipids

Patterns of the biosynthesis ofmajor metabolites of the oleaginous yeast Cryptococcus curvatus NRRL Y-1511 were investigated during cultivation on sugar-based media. When lactose or sucrose was employed as substrate under nitrogen-limited conditions, the yeast strain accumulated high quantities of intra-cellular total sugars (ITS) at the beginning of fermentation (up to 68% w/w), with ITS values progressively decreasing to 20%, w/w, at the end of the fermentation. Decrease in ITS content and consumption of extracellular lactose led to a subsequent rise in lipid accumulation, reaching 29.8% in dry cell weight at 80 g/L of initial lactose concentration. Lactose was a more favorable substrate for lipid production than sucrose. In nitrogen-excess conditions, ITS were produced in significant quantities despite the continuous presence of nitrogen in the medium. Growth on lactose was not followed by secretion of extra-cellular b-galactosidase. High quantities of extra-cellular invertase were observed during growth on sucrose. The composition of ITS was highly influenced by the sugar used as substrate. Cellular lipids contained mainly palmitic and to lesser extent linoleic and stearic acids. This is the first report in the literature that demonstrates the interplay between the biosynthesis of intra-cellular total sugars and lipid synthesis for oleaginous yeast strains.

Intracellular trafficking, localization, and mobilization of platelet-borne thiol isomerases

OBJECTIVE: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. APPROACH AND RESULTS: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13dJinx mice). CONCLUSIONS: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.