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PubMed1996

[Functional mapping of the motor and primary sensorial cortex using magnetic resonance techniques. I. Preliminary data].

Functional Magnetic Resonance Imaging (fMRI) techniques sensitive to the local changes in blood flow, volume and oxygenation accompanying neuronal activation are powerful tools to investigate the human brain function. Experiments were performed on 10 right-handed healthy volunteers (age range: 20-39 years), using a 1.5 T whole-body MRI system. Two oblique contiguous planes were investigated along the central sulcus of the left hemisphere. Functional images were acquired using a Gradient Echo sequence while the subjects repetitively performed sequential finger to thumb opposition movements of the right hand or mental imagery of a visual scene. Twelve images for each task were obtained over a 6-min experimental period; they were then analyzed with the software provided by the manufacturer. In all the subjects small areas were activated in both the precentral and postcentral gyrus, mean percentage signal increases during finger movement being 10.7% and 3.8%, respectively. These values are fairly higher than literature ones. However several factors, such as voxel volume, are involved in determining the measured signal increase during activation. Moreover, in most cases the software procedures provided with the MR equipment to analyze the functional images imply subjective choices. It is thus necessary to implement new software packages for the analysis of fMRI images to apply more appropriate statistical procedures and to obtain more homogeneous and objective final information.

Adult↗
PubMed2000

Body fluid regulation in micro-gravity differs from that on Earth: an overview.

Similar to the response to central hypervolemic conditions on Earth, the shift of blood volume from the legs to the upper part of the body in astronauts entering micro-gravity should, in accordance with the Henry-Gauer mechanism, mediate diuresis and natriuresis. However, fluid balance and kidney function experiments during various space missions resulted in the surprising observation that the responses qualitatively differ from those observed during simulations of hypervolemia on Earth. There is some evidence that the attenuated responses of the kidney while entering weightlessness, and also later during space flight, may be caused by augmented fluid distribution to extravascular compartments compared to conditions on Earth. A functional decoupling of the kidney may also contribute to the observation that renal responses during exposure to micro-gravity are consistently weaker than those during simulation experiments before space flight. Deficits in body mass after landing have always been interpreted as an indication of absolute fluid loss early during space missions. However, recent data suggest that body mass changes during space flight are rather the consequences of hypocaloric nutrition and can be overcome by improved nutrition schemes. Finally, sodium-retaining humoral systems are activated during space flight and may contribute to a new steady-state of metabolic balances with a pronounced increase in body sodium compared to respective conditions on Earth. A revision of the classical "micro-gravity fluid shift" scheme is required.

Body Fluids↗
PubMed2003

EphB receptors and ephrinB ligands: regulators of vascular assembly and homeostasis.

Eph receptors comprise the largest family of receptor tyrosine kinases consisting of eight EphA receptors (with five corresponding glycosyl-phosphatidyl-inositol-anchored ephrinA ligands) and six EphB receptors (with three corresponding transmembrane ephrinB ligands). Originally identified as neuronal pathfinding molecules, genetic loss of function experiments have identified EphB receptors and ephrinB ligands as crucial regulators of vascular assembly, orchestrating arteriovenous differentiation and boundary formation. Despite these clearly defined rate-limiting roles of the EphB/ephrinB system for developmental angiogenesis, the mechanisms of the functions of EphB receptors and ephrinB ligands in the cells of the vascular system are poorly understood. Moreover, little evidence can be found in the recent literature regarding complementary EphB and ephrinB expression patterns that occur in the vascular system and that may bring cells into juxtapositional contact to allow bi-directional signaling between neighboring cells. This review summarizes the current knowledge of the role of EphB receptors and ephrinB ligands during embryonic vascular assembly and discusses recent findings on EphB/ephrinB-mediated cellular functions pointing to the crucial role of the Eph/ephrin system in controlling vascular homeostasis in the adult.

Animals↗
PubMed1998

Cell proliferation in human prostatic smooth muscle cells involves the modulation of protein kinase C isozymes.

We have examined the role of protein kinase C in the regulation of foetal-calf serum-stimulated cell proliferation in human prostatic smooth muscle cells. The data showed that the proliferative effect to foetal-calf serum (10%, v/v) was partially inhibited by 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo (2,3-a) pyrrolo (3,4-c)-carbazole (Go-6976), a selective Ca2+-dependent protein kinase C inhibitor, suggesting that Ca2+-dependent protein kinase C isozymes might play roles in this proliferative regulation. Additionally, foetal-calf serum caused a significant translocation of protein kinase C-betaII and -epsilon from a cytosolic to a membrane distribution. These findings combined with the aforementioned functional experiments suggest that foetal-calf serum-stimulated cell proliferation might involve the activation of protein kinase C-betaII in human prostatic smooth muscle cells; however, the role of protein kinase C-epsilon in mediating cellular functions other than cell proliferation remains further investigation in these cells.

Carbazoles↗
PubMed1998

Interaction of the anxiogenic agent, RS-30199, with 5-HT1A receptors: modulation of sexual activity in the male rat.

RS-30199 has been shown previously to have atypical interactions at 5-HT1A receptors. RS-30199 and RS-64459, an analogue of buspirone with a buspirone side chain, were compared with the classic, partial agonist at 5-HT1A receptors, 8-hydroxy-2 (di-n-propylamino) tetralin (8-OH-DPAT) and buspirone. At human (h) 5-HT1A receptors in CHO cells, RS-30199-193 (racemate) and its enantiomers (-197, -198) inhibited [3H]-8-OH-DPAT binding (RS-30199-198, ki, 29.7 +/- 11.7 nM; RS-30199-197, ki, 74.1 +/- 11.7 nM) as did RS-64459 (ki, 24.9 +/- 6.0 nM), but RS-30199-197 and -198 were almost full agonists in a [35S]-GTPgammaS binding assay, whereas RS-64459 was a partial agonist, resembling buspirone and 8-OH-DPAT. RS-64459 and the enantiomers of RS-30199 had weaker affinity for 5-HT2C and 5-HT7 receptors. These compounds did not induce the 5-HT behavioural syndrome in male rats. However, in a model where naive male rats were introduced to estrogen-progesterone primed, sexually receptive female rats, RS-30199-197 (0.1, 1, 10 mg/kg, s.c.) had a profound inhibitory effect on sexual behaviour score. Neither buspirone nor 8-OH-DPAT reduced the sexual behaviour score. Unlike 8-OH-DPAT, which shortens intromission latency, RS-30199 prolonged intromission latency. RS-30199 (10 mg/kg s.c.) fully inhibited the facilitation of sexual behaviour caused by the alpha2-adrenoceptor antagonist, delequamine (0.1 mg/kg, p.o.). In contrast, RS-64459 (100, 250, 1000 and 4000 microg/kg, s.c.) failed to modify the sexual behaviour score and did not modify intromission latency. The differences between the effects of RS-30199 and RS-64459 in binding and functional experiments are supported by molecular models of the receptor-ligand interaction, where the compounds interact in different ways with the receptor; a model is proposed for the allosteric interaction of different agents with the receptor, resulting in different functional profiles. RS-30199 can be considered an atypical agonist at 5-HT1A receptors.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed2001

Crossinhibitory activities of Ngn1 and Math1 allow specification of distinct dorsal interneurons.

Distinct classes of neurons are generated from progenitor cells distributed in characteristic dorsoventral patterns in the developing spinal neural tube. We define restricted neural progenitor populations by the discrete, nonoverlapping expression of Ngn1, Math1, and Mash1. Crossinhibition between these bHLH factors is demonstrated and provides a mechanism for the generation of discrete bHLH expression domains. This precise control of bHLH factor expression is essential for proper neural development since as demonstrated in both loss- and gain-of-function experiments, expression of Math1 or Ngn1 in dorsal progenitor cells determines whether LH2A/B- or dorsal Lim1/2-expressing interneurons will develop. Together, the data suggest that although Math1 and Ngn1 appear to be redundant with respect to neurogenesis, they have distinct functions in specifying neuronal subtype in the dorsal neural tube.

Animals↗
PubMed2003

Modifications to the N-terminus but not the C-terminus of calcitonin gene-related peptide(8-37) produce antagonists with increased affinity.

Seventeen novel analogues of human calcitonin gene-related peptide(8-37) (hCGRP(8-37)) were synthesized by solid-phase methods and purified to apparent homogeneity by semipreparative cation exchange and/or reversed-phase high-performance liquid chromatography. The C-terminal Phe was replaced by Gly, cyclohexylalanine (Cha), Tyr, all four isomers of beta-methylphenylalanine (beta-MePhe), and l- and d-tetrahydroisoquinoline carboxylic acid (Tic), resulting in analogues 3-11. For the synthesis of the beta-MePhe-containing analogues 6-9, crystallization was used to separate a mixture of all four isomers of beta-MePhe into the erythro pair of enantiomers (2S,3S, 2R,3R) and the threo pair of enantiomers (2S,3R, 2R,3S), which were then converted to Fmoc derivatives and used in two separate syntheses. Two diastereomeric peptides were obtained from each synthesis and were separated by RP-HPLC to yield enantiomerically pure 6-9. Substitution of Tyr for Phe caused no change in binding affinity at CGRP receptors. All other substitutions for Phe resulted in substantial reductions in binding affinity. Indeed, no binding was observed for analogues 7, 9, and 11, all of which contained a d-amino acid residue in the C-terminal position, and the binding affinities of the remaining analogues were >10-fold lower than that of h-alpha-CGRP(8-37). These data suggest that a conformationally flexible phenyl ring in the C-terminal position of h-alpha-CGRP(8-37) is preferred for high-affinity binding to CGRP receptors. Acetylation, benzoylation, and benzylation of the N-termini of h-alpha-CGRP(8-37) and h-beta-CGRP(8-37) produced analogues 12-14 and 16-18, respectively. A byproduct was isolated by RP-HPLC from the resin-cleaved crude product of each benzylated analogue, which was characterized as the dibenzylated derivative of h-alpha-CGRP(8-37) and h-beta-CGRP(8-37) (analogues 15 and 19, respectively). Amino acid analysis and (1)H NMR showed that the second benzyl group was located on the C4 carbon of the imidazole ring of His(10). Radioligand binding experiments showed that derivatizing the N-termini substantially increased binding affinities at CGRP receptors. The benzoylated and dibenzylated derivatives had the highest affinities, which were approximately 50-fold greater than those of h-alpha-CGRP(8-37). Functional experiments confirmed that the N-terminally derivatized analogues of h-alpha-CGRP(8-37) are antagonists that are more potent than h-alpha-CGRP(8-37). In conclusion, these studies underscore the importance of Phe(37) of h-alpha-CGRP(8-37) for binding to CGRP receptors and have identified the N-terminus and His(10) as two positions that can be used for the design of antagonists with increased affinity for CGRP receptors.

Animals↗
PubMed2003

Identification of a surface for binding to the GDNF-GFR alpha 1 complex in the first cadherin-like domain of RET.

The RET receptor tyrosine kinase is activated by binding to a ligand complex formed by a member of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors bound to its cognate GDNF-family receptor-alpha (GFR alpha) glycosylphosphatidylinositol-linked co-receptor. Molecular modeling studies of the extracellular domain of RET (RETECD) have revealed the existence of four cadherin-like domains (CLD1-4) followed by a cysteine-rich domain. Cross-linking experiments have indicated that the RETECD makes direct contacts with both the GDNF ligand and GFR alpha 1 molecule in the complex, although it has low or no detectable affinity for either component alone. We have exploited sequence and functional divergences between the ectodomains of mammalian and amphibian RET molecules to map binding determinants in the human RETECD responsible for its interaction with the GDNF-GFR alpha 1 complex by homologue-scanning mutagenesis. We found that Xenopus RETECD was unable to bind to GDNF-GFR alpha-1 or neurturin (NTN)-GFR alpha-2 complexes of mammalian origin. However, a chimeric molecule containing CLD1, -2, and -3 from human RETECD, but neither domain alone, had similar binding activity as compared with wild type human RETECD, suggesting the existence of an extended ligand binding surface within the three N-terminal cadherin-like domains of human RETECD. Subsequent loss-of-function experiments at higher resolution identified three small subsets of residues, mapping on the same face of the molecular model of RET CLD1, that were required for the interaction of human RETECD with the GDNF-GFR alpha 1 complex. Additional experiments demonstrated that N-linked glycosylation of human RETECD was not required for ligand binding. Based on these observations, we propose a model for the assembly and architecture of the GDNF-GFR alpha 1-RET complex.

Amino Acid Sequence↗
PubMed2005

Identification of core functional region of murine IL-4 using peptide phage display and molecular modeling.

Murine IL-4 is a pleiotropic cytokine with undefined core functional region for eliciting downstream signaling. We used molecular modeling to predict the binding sites recognized by an anti-IL-4-neutralizing mAb (11B.11) and peptide phage display to delineate their makeup. The results of these approaches were confirmed by site-directed mutagenesis analysis. The results suggest that the amino acid residues spanning from 79 to 86 (QRLFRAFR) on IL-4 are of the major binding site for 11B.11. Furthermore, the functional experiments demonstrate that the residues R80, R83 and R86, which are located in the helix C of murine IL-4, play a crucial role in binding to the IL-4R alpha-chain. Taken together, a new core functional region of murine IL-4 is identified, which provides new insight into the interaction between IL-4 and IL-4Ralpha. In addition, the results demonstrate that 11B.11 binds to a core functional region of murine IL-4, which prevents this cytokine from interacting with its cognate receptor.

Amino Acid Substitution↗
PubMed1992

Pain assessment in spinal cord injury.

Pain management is a serious problem for individuals with spinal cord injury (SCI). Recent developments in pain assessment indicate that multiaxial approaches, assessing medical, psychosocial, and behavioral/functional dimensions, are necessary to measure adequately the impact of chronic pain. The application of this multiaxial system to persons with SCI and chronic pain is presented. A review of the literature indicates that the psychometric properties and test utility for most pain measures have not been established for persons with SCI. The assessment task is further confounded by the functional limitations and psychosocial impairments that may accompany SCI. Recommendations are made for adapting established pain measures for use with SCI individuals. The choice of assessment tools for these patients is guided by the multidimensional nature of the pain experience, functional limitations, and the goals of treatment.

Humans↗
PubMed2005

Normal immune system development in mice lacking the Deltex-1 RING finger domain.

The Notch signaling pathway controls several cell fate decisions during lymphocyte development, from T-cell lineage commitment to the peripheral differentiation of B and T lymphocytes. Deltex-1 is a RING finger ubiquitin ligase which is conserved from Drosophila to humans and has been proposed to be a regulator of Notch signaling. Its pattern of lymphoid expression as well as gain-of-function experiments suggest that Deltex-1 regulates both B-cell lineage and splenic marginal-zone B-cell commitment. Deltex-1 was also found to be highly expressed in germinal-center B cells. To investigate the physiological function of Deltex-1, we generated a mouse strain lacking the Deltex-1 RING finger domain, which is essential for its ubiquitin ligase activity. Deltex-1(Delta/Delta) mice were viable and fertile. A detailed histological analysis did not reveal any defects in major organs. T- and B-cell development was normal, as were humoral responses against T-dependent and T-independent antigens. These data indicate that the Deltex-1 ubiquitin ligase activity is dispensable for mouse development and immune function. Possible compensatory mechanisms, in particular those from a fourth Deltex gene identified during the course of this study, are also discussed.

Amino Acid Sequence↗
PubMed2006

Gene expression profiling of breast cancer cells in response to gemcitabine: NF-kappaB pathway activation as a potential mechanism of resistance.

Gemcitabine is a nucleoside analog with clinical relevance in the treatment of several solid tumors, including breast carcinoma. In spite of its cytotoxic effect, clinical efficacy is impaired by the development of resistance. We performed gene expression analysis to shed light into the molecular mechanism of action of this drug in two breast cancer cell lines. Activation of genes related with cell cycle, cell growth and apoptosis (BNIP3L, CCNG2, DDIT4, TGFB2, TP53BP1, TP53INP1, and VEGF) was the main finding in the p53-wild type cell line MCF7, while the p53-non-functional cell line MDA-MB-231 was characterized by the regulation of NF-kappaB target genes (BIRC3, CXCL1/GRO1, IRAK2, TNF, TNFAIP and TRAF1). Genes consistently induced (ATF3, CCNG2, CDKN1A, EGR1, INSIG1, and MAF) or repressed (CCND1 and VGF) in both cell lines, were also found after gemcitabine treatment. In addition, MDA-MB-231 cells showed a higher basal and induced NF-kappaB transcriptional activity after treatment with gemcitabine. In comparison with gemcitabine, gene expression after 5-fluorouracil treatment showed essentially different profiles in both cell lines. This, in spite of using equitoxic concentrations producing similar effects on cell cycle. NF-kappaB transcriptional activity in MDA-MB-231 cells was dependent on IkappaB-alpha phosphorylation, as shown by functional experiments using the specific inhibitor BAY11-7082. Moreover, immunohistochemical analysis of clinical samples of breast carcinoma further validated the induction of NF-kappaB expression and IkappaB down-regulation upon neoadjuvant gemcitabine treatment. Thus, gene expression patterns, in vitro functional studies and analysis of tissue samples are in agreement with a role for NF-kappaB pathway in gemcitabine response. Together with the reported role for NF-kappaB in the induction of resistance to chemotherapy, our data gives support to clinical strategies combining gemcitabine with NF-kappaB inhibitors in breast cancer.

Antimetabolites, Antineoplastic↗
PubMed1985

Differential labeling of platelet alpha 2 adrenoceptors by 3H dihydroergocryptine and 3H yohimbine in patients with myeloproliferative disorders.

Platelet alpha adrenoceptor status was examined using the radioligands 3H-yohimbine (3H-YOH) and 3H-dihydroergocryptine (3H-DHE) in 14 patients with myeloproliferative disorder (MPD) and 10 normal controls. Platelets from normal controls and MPD patients sensitive to adrenaline induced aggregation exhibited approximately 50% more binding sites identified by 3H-DHE than 3H-YOH, whereas MPD platelets insensitive to adrenaline showed selective loss of these 'extra' 3H-DHE sites. In functional studies after 30 minutes preincubation with the unlabelled antagonists, DHE was more potent than YOH at inhibiting adrenaline induced aggregation in normal platelets. In addition, the affinity constant for DHE was virtually identical in binding and functional experiments, whereas for YOH the affinity constant for binding was approximately 10 fold more potent than that for aggregation. These results suggest that the alpha adrenoceptor binding site on human platelets labelled by 3H-DHE may be of more functional relevance than that labelled by 3H-YOH alone.

Blood Platelets↗
PubMed2005

Homeotic selector genes control the patterning of seven-up expressing cells in the Drosophila dorsal vessel.

The linear cardiac tube of Drosophila, the dorsal vessel, is an important model organ for the study of cardiac specification and patterning in vertebrates. In Drosophila, the Hox segmentation gene abdominal-A (abd-A) is required for the specification of a functionally distinct heart region at the posterior of the dorsal vessel, from which blood is pumped anteriorly through a tube termed the aorta. Since we have previously shown that the posterior part of the aorta is specified during embryogenesis to form the adult heart during metamorphosis, we determined if the embryonic aorta is also patterned by the function of Hox segmentation genes. Using gain- and loss-of-function experiments, we demonstrate that the three Hox genes expressed in the posterior aorta and heart are sufficient to confer heart or posterior aorta fate throughout the dorsal vessel. Additionally, we demonstrate that Ultrabithorax and abd-A, but not Antennapedia, function to control cell number in the dorsal vessel. These studies add robustness to the model that homeotic selector genes pattern the Drosophila dorsal vessel, and further extend our understanding of how the cardiac tube is patterned in animal models.

Animals↗
PubMed2002

Cadherins and the tumour progression: is it all in a switch?

Progression to tumour malignancy involves changes in a tumour cell's capabilities to adhere and communicate with neighboring cells and with its extracellular environment. Correlation studies in human cancer specimen and functional experiments with cultured tumour cells and transgenic mouse models have indicated that the loss of the cell adhesion molecule E-cadherin is causally involved in the formation of epithelial cancers (carcinomas). More recently, it has been observed that the function of E-cadherin is replaced or overruled by the expression of mesenchymal cadherins, such as N-cadherin. Although the functional implication of such a 'cadherin switch' remains to be elucidated, recent experimental results demonstrating an interaction of cadherins with tyrosine kinase receptors suggest that changes in cadherin expression may not only modulate tumour cell adhesion but also affect signal transduction and, hence, the malignant phenotype.

Animals↗
PubMed1993

MHC class I/peptide interactions: binding specificity and kinetics.

Recent developments in the preparation of soluble analogues of the major histocompatibility complex (MHC) class I molecules as well as in the application of real time biosensor technology have permitted the direct analysis of the binding of MHC class I molecules to antigenic peptides. Using synthetic peptide analogues with cysteine substitutions at appropriate positions, peptides can be immobilized on a dextran-modified gold biosensor surface with a specific spatial orientation. A full set of such substituted peptides (known as 'pepsicles', as they are peptides on a stick) representing antigenic or self peptides can be used in the functional mapping of the MHC class I peptide binding site. Scans of sets of peptide analogues reveal that some amino acid side chains of the peptide are critical to stable binding to the MHC molecule, while others are not. This is consistent with functional experiments using substituted peptides and three-dimensional molecular models of MHC/peptide complexes. Detailed analysis of the kinetic dissociation rates (kd) of the MHC molecules from the specifically coupled solid phase peptides reveals that the stability of the complex is a function of the particular peptide, its coupling position, and the MHC molecule. Measured kd values for antigenic peptide/class I interactions at 25 degrees C are in the range of ca 10(-4)-10(-6)/s. Biosensor methodology for the analysis of the binding of MHC class I molecules to solid-phase peptides using real time surface plasmon resonance offers a rational approach to the general analysis of protein/peptide interactions.

Amino Acid Sequence↗
PubMed1999

WB 4101-related compounds. 2. Role of the ethylene chain separating amine and phenoxy units on the affinity for alpha(1)-adrenoreceptor subtypes and 5-HT(1A) receptors.

WB 4101 (1)-related benzodioxanes were synthesized by replacing the ethylene chain separating the amine and the phenoxy units of 1 with a cyclopentanol moiety, a feature of 6, 7-dihydro-5-[[(cis-2-hydroxy-trans-3-phenoxycyclopentyl)amino]meth yl] -2-methylbenzo[b]thiophen-4(5H)-one that was reported to display an intriguing selectivity profile at alpha(1)-adrenoreceptors. This synthesis strategy led to 4 out of 16 possible stereoisomers, which were isolated in the case of (-)-3, (+)-3, (-)-4, and (+)-4 and whose absolute configuration was assigned using a chiral building block for the synthesis of (-)-3 starting from (+)-(2R)-2, 3-dihydro-1,4-benzodioxine-2-carboxylic acid ((+)-9) and (1S,2S, 5S)-2-amino-5-phenoxycyclopentan-1-ol ((+)-10). The aim of this project was to further investigate whether it is possible to differentiate between these compounds with respect to their affinity for alpha(1)-adrenoreceptor subtypes and the affinity for 5-HT(1A) receptors, as 1 binds with high affinity at both receptor systems. The biological profiles of reported compounds at alpha(1)-adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha(1A)), spleen (alpha(1B)), and aorta (alpha(1D)) and by binding assays in CHO and HeLa cells membranes expressing the human cloned alpha(1)-adrenoreceptor subtypes and 5-HT(1A) receptors, respectively. Furthermore, the functional activity of (-)-3, (+)-3, (-)-4, and (+)-4 toward 5-HT(1A) receptors was evaluated by determining the induced stimulation of [(35)S]GTPgammaS binding in cell membranes from HeLa cells transfected with human cloned 5-HT(1A) receptors. The configuration of the cyclopentane unit determined the affinity profile: a 1R configuration, as in (+)-3 and (-)-4, conferred higher affinity at alpha(1)-adrenoreceptors, whereas a 1S configuration, as in (-)-3 and (+)-4, produced higher affinity for 5-HT(1A) receptors. For the enantiomers (+)-4 and (-)-4 also a remarkable selectivity was achieved. Functionally, the stereoisomers displayed a similar alpha(1)-selectivity profile, that is alpha(1D) > alpha(1B) > alpha(1A), which is different from that exhibited by the reference compound 1. The epimers (-)-3 and (+)-4 proved to be agonists at the 5-HT(1A) receptors, with a potency comparable to that of 5-hydroxytryptamine.

Adrenergic alpha-Agonists↗
PubMed2005

Calcium-independent phospholipase A2 modulates cytosolic oxidant activity and contractile function in murine skeletal muscle cells.

Phospholipase A2 (PLA2) activity supports production of reactive oxygen species (ROS) by mammalian cells. In skeletal muscle, endogenous ROS modulate the force of muscle contraction. We tested the hypothesis that skeletal muscle cells constitutively express the calcium-independent PLA2 (iPLA2) isoform and that iPLA2 modulates both cytosolic oxidant activity and contractile function. Experiments utilized differentiated C2C12 myotubes and a panel of striated muscles isolated from adult mice. Muscle preparations were processed for measurement of mRNA by real-time PCR, protein by immunoblot, cytosolic oxidant activity by the dichlorofluorescein oxidation assay, and contractile function by in vitro testing. We found that iPLA2 was constitutively expressed by all muscles tested (myotubes, diaphragm, soleus, extensor digitorum longus, gastrocnemius, heart) and that mRNA and protein levels were generally similar among muscles. Selective iPLA2 blockade by use of bromoenol lactone (10 microM) decreased cytosolic oxidant activity in myotubes and intact soleus muscle fibers. iPLA2 blockade also inhibited contractile function of unfatigued soleus muscles, shifting the force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2 modulates oxidant activity in skeletal muscle fibers by supporting ROS production, thereby influencing contractile properties and fatigue characteristics.

Animals↗