Rapid negative inotropic effect induced by TNF-α in rat heart perfused related to PKC activation.

Myocardial depression, frequently observed in septic shock, is mediated by circulating molecules such as cytokines. TNF-α appears to be the most important pro-inflammatory cytokine released during the early phase of a septic shock. It was previously shown that TNF-α had a negative inotropic effect on myocardium. Now, the aim of this study was to investigate the effects of the activation of PKC by TNF-α on heart function, and to determine if this cytokine could induce a decrease of membrane excitability. Isolated rat hearts (n = 6) were perfused with Tyrode solution containing TNF-α at 20 ng/ml during 30 min by using a Langendorff technique. Expressions of PKC-α and PKC-ε were analysed by western blot on membrane and cytosol proteins extracted from ventricular myocardium. Patch clamp was performed on freshly isolated cardiomyocytes (n = 8). Compared to control situation, 30 min of TNF-α perfusion led to cardiac dysfunction with a decrease of the heart rate (-83%), the force (-20%) and speed of relaxation (-18%) and the coronary flow (-25%). This is associated with an activation and a membrane targeting of both PKC-α and PKC-ε isoforms in ventricle with respectively +123% and +54% compared to control hearts. Nevertheless, TNF-α had no significant effect on voltage-gated sodium current (109.0%+/- 12.5) after addition of the cytokine when compared to control. These results showed that TNF-α had a negative inotropic effect on the isolated rat heart and can induce PKC activation leading to an impaired contractility of the heart. However the early heart dysfunction induced by the cytokine was not associated to a decrease of cardiomyocytes membrane excitability as it has been evidenced in skeletal muscle fibres.

A re-innervated in vitro skin model of non-histaminergic itch and skin neurogenic inflammation: PAR2-, TRPV1- and TRPA1-agonist induced functionality.

Background: Skin, and epidermis, is innervated by sensory nerve fibres. Interactions between them and signal transduction are only partially elucidated in physiological/pathological conditions, especially in pruritus. Objectives: To study the mechanisms involved in pruritus in vitro, we developed a skin explant model re-innervated by sensory neurons. Methods: This model is based on the co-culture of human skin explants and sensory neurons from dorsal root ganglia of rats. Innervation and the expression of protease activated receptor 2 (PAR2), transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin one (TRPA1) was analysed by immunostaining. The response of the model to TRPV1, PAR2 and TRPA1 agonists was analysed by patch-clamp, qPCR and enzyme-linked immunosorbent assay. Results: After 5 days of re-innervating nerve fibres was evidenced in the epidermis. Re-innervation was correlated with decrease of epidermal thickness and the number of apoptotic cells in the tissue. The major actors of non-histaminergic itch (PAR-2, thymic stromal lymphopoietin [TSLP], TSLP-R, TRPA1 and TRPV1) were expressed in neurons and/or epidermal cells of skin explants. After topical exposure of TRPV1-(Capsaicin), TRPA1-(Polygodial) and PAR2-agonist (SLIGKV-NH2) activation of reinnervating neurons could be shown in patch-clamp analysis. The release of TSLP was increased with capsaicin or SLIGKV but decreased with polygodial. Release of CGRP was increased by capsaicin and polygodial but decreased with SLIGKV. Activation by SLIGKV showed a decrease of VEGF; polygodial induced an increase of TSLP, Tumour necrosis factor (TNF) and nerve growth factor and capsaicin lead to a decrease of sema3 and TNF expression. Conclusion: The present model is suitable for studying itch and neurogenic inflammation pathways in vitro. We observed that activation of TRPV1, TRPA1 and PAR-2 leads to different response profiles in re-innervated skin explants.

Sodium channel Na(V)1.5 expression is enhanced in cultured adult rat skeletal muscle fibers.

This study analyzes changes in the distribution, electrophysiological properties, and proteic composition of voltage-gated sodium channels (Na(V)) in cultured adult rat skeletal muscle fibers. Patch clamp and molecular biology techniques were carried out in flexor digitorum brevis (FDB) adult rat skeletal muscle fibers maintained in vitro after cell dissociation with collagenase. After 4 days of culture, an increase of the Na(V)1.5 channel type was observed. This was confirmed by an increase in TTX-resistant channels and by Western blot test. These channels exhibited increased activation time constant (tau(m)) and reduced conductance, similar to what has been observed in denervated muscles in vivo, where the density of Na(V)1.5 was increasing progressively after denervation. By real-time polymerase chain reaction, we found that the expression of beta subunits was also modified, but only after 7 days of culture: increase in beta(1) without beta(4) modifications. beta(1) subunit is known to induce a negative shift of the inactivation curve, thus reducing current amplitude and duration. At day 7, tau(h) was back to normal and tau(m) still increased, in agreement with a decrease in sodium current and conductance at day 4 and normalization at day 7. Our model is a useful tool to study the effects of denervation in adult muscle fibers in vitro and the expression of sodium channels. Our data evidenced an increase in Na(V)1.5 channels and the involvement of beta subunits in the regulation of sodium current and fiber excitability.

Mechanisms of the sensory effects of tacrolimus on the skin.

BACKGROUND: Tacrolimus is an immunosuppressant drug currently used for the treatment of atopic dermatitis and pruritus. This topical therapy is effective and safe, but transient burning, stinging and itch are frequently reported. OBJECTIVES: To understand the mechanisms underlying these burning sensations. METHODS: We examined the impact of tacrolimus on substance P (SP) release in an in vitro model of cutaneous neurogenic inflammation. Because phosphorylation of TRPV1 (transient receptor potential subtype vanilloid 1) plays a role in the induction of pain, we investigated whether tacrolimus regulates the phosphorylation state of TRPV1. Finally, we used a macropatch to evaluate the impact of tacrolimus on voltage-gated calcium currents of sensory neurons. RESULTS: Tacrolimus was able to induce initial SP release by extracellular calcium influx and inhibited SP release induced by capsaicin after 1, 24 and 72 h of pretreatment. Analysis of TRPV1 phosphorylation by Western blot confirmed the capacity of tacrolimus to favour phosphorylation. An electrophysiological study showed inhibitory effects on calcium currents. CONCLUSIONS: The efficacy of tacrolimus in pruritus, as well as the sensory side-effects, could be explained by a direct effect on neurons through an effect on calcineurin, possibly by a desensitization of TRPV1 and calcium currents through the PIP(2) regulation pathway.

Differences in sodium voltage-gated channel properties according to myosin heavy chain isoform expression in single muscle fibres.

The myosin heavy chain (MHC) isoform determines the characteristics and shortening velocity of muscle fibres. The functional properties of the muscle fibre are also conditioned by its membrane excitability through the electrophysiological properties of sodium voltage-gated channels. Macropatch-clamp is used to study sodium channels in fibres from peroneus longus (PL) and soleus (Sol) muscles (Wistar rats, n = 8). After patch-clamp recordings, single fibres are identified by SDS-PAGE electrophoresis according to their myosin heavy chain isoform (slow type I and the three fast types IIa, IIx, IIb). Characteristics of sodium currents are compared (Student's t test) between fibres exhibiting only one MHC isoform. Four MHC isoforms are identified in PL and only type I in Sol single fibres. In PL, maximal sodium current (I(max)), maximal sodium conductance (g(Na,max)) and time constants of activation and inactivation ((m) and (h)) increase according to the scheme I-->IIa-->IIx-->IIb (P < 0.05). (m) values related to sodium channel type and/or function, are similar in Sol I and PL IIb fibres (P = 0.97) despite different contractile properties. The voltage dependence of activation (V(a,1/2)) shows a shift towards positive potentials from Sol type I to IIa, IIx and finally IIb fibres from PL (P < 0.05). These data are consistent with the earlier recruitment of slow fibres in a fast-mixed muscle like PL, while slow fibres of postural muscle such as soleus could be recruited in the same ways as IIb fibres in a fast muscle.

A possible calcium binding site in animal lectins: a 1H-NMR study of the interaction between lanthanides and a synthetic peptide from a highly conserved domain of Pleurodeles lectin.

1H-NMR techniques have been used to study the metal binding properties of a synthetic peptide of 15 amino acids corresponding to a highly conserved domain of Pleurodeles lectin. The addition of lanthanum chloride or praseodymium chloride in a peptide solution induces some conformational changes as displayed by several concerted variations of peptide resonances. The Ln3+ concentration dependence of the chemical shifts was used to calculate the Ln3+ binding constants. The dissociation constants of 95 microM and 280 microM were found for La3+ and Pr3+, respectively.

Organization of a rainbow trout estrogen receptor gene.

The complete coding region of the estrogen receptor gene was isolated from a rainbow trout genomic library. This gene is divided into ten exons spanning at least 30 kb of genomic DNA. With two exceptions, intron positions are identical to those of the human estrogen receptor gene. The 5' end of the gene, including 1.7 kb of the promoter region, was sequenced. This region exhibits several putative regulatory elements. Localization of a potential estrogen responsive element to the first exon suggests that this gene is autoregulated. This 5' end region was also shown to be able to drive the expression of a CAT reporter gene in Xenopus laevis oocytes.

Cloning of the Xenopus laevis cdk2 promoter and functional analysis in oocytes and during early development.

CDK2 (cyclin-dependent kinase 2) is a serine/threonine kinase which is involved in regulating S-phase entry in higher eukaryotes. To investigate the transcriptional control of this gene, a 13-kb Xenopus laevis genomic clone containing the 5' flanking sequences was isolated. A 2.7-kb fragment containing the promoter region was sequenced and the transcription start point (tsp) was determined by primer extension. Several putative regulatory elements, such as the E2F-binding site, Y box and octamer-binding site, were localized in this region, but no TATA box was found. When fused to cat, a reporter gene encoding chloramphenicol acetyltransferase, the 5' flanking sequences were shown to function in oocytes and an enhancer activity was found in this region. During early embryogenesis, cdk2 promoter activity was tested and de novo transcription was detected at the mid-blastula transition.

Characterization of vitellogenin from rainbow trout (Oncorhynchus mykiss).

The nucleotide sequence of the vitellogenin cDNA from the rainbow trout Oncorhynchus mykiss was determined. Analysis of the deduced amino acid sequence (1659 residues) places the lipovitellin I, phosvitin and lipovitellin II domains between amino acids 16 to 1088, 1089 to 1145 and 1146 to 1659, respectively. The general structure is similar to other vertebrate vitellogenins except for the serine rich phosvitin domain which is the shortest identified so far in vertebrates (57 amino acids), being 2 to 4 times smaller than in other species. Sequence comparisons between vertebrate and invertebrate vitellogenins as well as with distantly related proteins allowed to identify two short amino acid motifs particularly well conserved, RGILN and TCGLCG in lipovitellin I and II domains, respectively, and strongly suggest that the lipovitellin II domain is involved in protein interactions via disulfide bridge formation.

Structure of a fish (Oncorhynchus mykiss) vitellogenin gene and its evolutionary implication.

In this paper we describe the first complete structure of a fish vitellogenin gene. A 22 kb genomic region from rainbow trout (Oncorhynchus mykiss) was cloned and analysed. This region was shown to contain two tandemly arranged vitellogenin genes. Both genes are 98.7% similar, indicating that they result from a recent local duplication. The complete sequence encoding one of the two genes was determined and the gene organization was established. The gene is 10.3 kb long and has 34 exons, it lacks one exon compared to amphibian and avian vitellogenin genes. Exons 22 and 23 of the Xenopus and chicken genes were shown to be merged into a single exon in the trout genome. Other splicing sites appeared highly conserved between the three vertebrate genes. In contrast, little similarity between invertebrate and vertebrate vitellogenin genes was observed with respect to the number and organization of introns. The comparison of 17 independent invertebrate splicing sites with the 34 vertebrate sites indicated that a few sites are probably ancient. However, most of the splicing junctions compared appeared unrelated. Results suggest that vitellogenin genes have been reshaped through multiple insertions and deletions of intervening sequences during evolution.

Two messenger RNAs and five isoforms for Po66-CBP, a galectin-8 homolog in a human lung carcinoma cell line.

Galectins are animal proteins which specifically bind beta-D-galactoside residues and their specific cellular function is not yet clearly established. However, these proteins seem to play a role in neoplastic transformations. Po66 is a murine monoclonal antibody directed against a protein from human lung carcinoma, Po66 Carbohydrate-Binding-Protein (Po66-CBP), which belongs to the galectin-8 family. Our results show that the Po66-CBP gene generates five transcripts by alternative splicing, which could give rise to five proteins: two proteins belong to the tandemly repeated galectin family and three belong to the single carbohydrate recognition domain galectins. All these proteins are encoded by a unique gene located in 1q42. Experiments carried out by reverse transcriptase-polymerase chain reaction show that the levels of expression of these five galectin-8 isoforms are variable during the culture time in SK-MES-1, a human lung squamous carcinoma cell line. Cancer Genome Anatomy Project database analysis confirms the presence of Po66-CBP in lung cancer and its absence in healthy lung.

Galectin-8: a complex sub-family of galectins (Review).

Galectins are animal lectins, that can specifically bind beta-galactosides. Twelve galectins have been described in vertebrates, belonging to three different groups: prototype, tandem-repeat and chimeric. These proteins seem to be involved in cellular interactions and neoplastic transformations. We present an overview of a particular galectin member: galectin-8. This galectin, which has been intensively studied over the last six years, presents a particular type of gene regulation. It is widely expressed in tumoral tissues and seems to be involved in integrin-like cell interactions. Studies show that the LGALS8 gene encodes for almost seven mRNAs by alternative splicing pathways and various polyadenylation sites. These mRNAs could encode for six isoforms of galectin-8, of which three belong to the tandem-repeat galectin group (with two carbohydrate binding domains) and the three others to the prototype group (one carbohydrate binding domain). All these isoforms seem to be differentially expressed in various tumoral cells. This untypical galectin-8 subfamily seems to have a complex expression regulation, that could be involved in cancer phenomena.

Purification of a tumoral marker recognized by monoclonal antibody Po66 and associated with human lung squamous cell carcinoma.

Monoclonal antibody (MAb) Po66, a murine IgG1, was raised by immunization against human lung squamous cell carcinoma. When injected intravenously, Po66 showed prolonged retention in the tumor. It recognized an intracellular antigen. The human lung squamous carcinoma cell line SK-MES-1 expresses the antigen recognized by MAb Po66 and was used as a source of biological material for its purification. The SK-MES-1 cell line was labeled in culture with [35S]methionine and its lysate was immunoprecipitated with Po66 immobilized on Protein G-Sepharose. The precipitate contained three proteins (47, 50 and 69 kDa) absent in the controls. The 69 kDa polypeptide was further purified by anion exchange and immunoaffinity chromatographies. To date, no other tumor marker expressed in non-small cell lung cancer with these characteristics has been described and as such this marker is interesting for future use in immunotherapy and in diagnosis.

Restriction fragment length polymorphism analysis of endogenous avian leukosis viral loci: determination of frequencies in commercial broiler lines.

Four component lines of a commercial broiler stock were analyzed by restriction fragment length polymorphism (RFLP) analysis, for the presence of endogenous avian leukosis virus loci. The resulting RFLP patterns of endogenous virus (ev) loci for the four strains were further characterized in terms of number of loci per animal and frequency. Loci were also analyzed within limits for major structural alterations and deletions to discern whether certain loci were similar to previously identified loci in White Leghorn layer birds. The ev RFLP patterns for these broiler lines were found to be highly complex and contained many loci unreported in White Leghorn layer birds.

The endogenous retroviral ev21 locus in commercial chicken lines and its relationship with the slow-feathering phenotype (K).

Provirus ev21 was found in both K- and k(+)-feathering Rhode Island Red commercial layers. Probe EV21-int revealed the presence of two distinct but similar regions, US (unoccupied site) and OS (occupied site). Restriction analysis showed that these regions had at least 19 kb structural homology but were distinguishable by ev21 proviral sequences, OS, and possibly three polymorphics US. The loci OS and US were both located on Chromosome Z. The k(+)-feathering birds were found to have only one site (either OS or US) per individual Z chromosome, whereas K-feathering birds had at least one Z chromosome with both regions in cis configuration. It has been possible to show that the reversion to the k(+)-feathering phenotype is accompanied by the loss of either a US or OS region that disrupts the cis configuration in K-feathering birds.

[ICU acquired neuromyopathy]. / La neuromyopathie acquise en réanimation.

ICU acquired neuromyopathy (IANM) is the most frequent neurological pathology observed in ICU. Nerve and muscle defects are merged with neuromuscular junction abnormalities. Its physiopathology is complex. The aim is probably the redistribution of nutriments and metabolism towards defense against sepsis. The main risk factors are sepsis, its severity and its duration of evolution. IANM is usually diagnosed in view of difficulties in weaning from mechanical ventilation, but electrophysiology may allow an earlier diagnosis. There is no curative therapy, but early treatment of sepsis, glycemic control as well as early physiotherapy may decrease its incidence. The outcomes of IANM are an increase in morbi-mortality and possibly long-lasting neuromuscular abnormalities as far as tetraplegia.

Effects of lactate on the voltage-gated sodium channels of rat skeletal muscle: modulating current opinion.

During muscle contraction, lactate production and translocation across the membrane increase. While it has recently been shown that lactate anion acts on chloride channel, less is known regarding a potential effect on the voltage-gated sodium channel (Na(v)) of skeletal muscle. The electrophysiological properties of muscle Na(v) were studied in the absence and presence of lactate (10 mM) by using the macropatch-clamp method in dissociated fibers from rat peroneus longus (PL). Lactate in the external medium (petri dish + pipette) increases the maximal sodium current, while the voltage dependence of activation and fast inactivation are shifted toward the hyperpolarized potentials. Lactate induces a leftward shift in the relationship between the kinetic parameters and the imposed potentials, resulting in an earlier recruitment of muscle Na(v). In addition, lactate significantly decreases the time constant of activation at voltages more negative than -10 mV, corresponding to an acceleration of Na(v) activation. The slow inactivation process is decreased by lactate, corresponding to an enhancement in the number of excitable Na(v). In an additional series of experiments, lactate (10 mM) was only added to the petri dish, while the pipette remained sealed on the membrane area. With this approach, the electrophysiological properties of Na(v) were unaffected by lactate compared with the control condition. Altogether, these data indicate that lactate modulates muscle Na(v) properties by an extracellular pathway. These effects are consistent with an enhancement in excitability, providing new insights into the role of lactate in muscle physiology.

Establishment of functional primary cultures of heart cells from the clam Ruditapes decussatus.

Heart cells from the clam Ruditapes decussatus were routinely cultured with a high level of reproducibility in sea water based medium. Three cell types attached to the plastic after 2 days and could be maintained in vitro for at least 1 month: epithelial-like cells, round cells and fibroblastic cells. Fibroblastic cells were identified as functional cardiomyocytes due to their spontaneous beating, their ultrastructural characteristics and their reactivity with antibodies against sarcomeric α-actinin, sarcomeric tropomyosin, myosin and troponin T-C. Patch clamp measurements allowed the identification of ionic currents characteristic of cardiomyocytes: a delayed potassium current (I (K slow)) strongly suppressed (95%) by tetraethylammonium (1 mM), a fast inactivating potassium current (I (K fast)) inhibited (50%) by 4 amino-pyridine at 1 mM and, at a lower level (34%) by TEA, a calcium dependent potassium current (I (KCa)) activated by strong depolarization. Three inward voltage activated currents were also characterized in some cardiomyocytes: L-type calcium current (I (Ca)) inhibited by verapamil at 5 × 10(-4) M, T-type Ca(2+) current, rapidly activated and inactivated, and sodium current (I (Na)) observed in only a few cells after strong hyperpolarization. These two currents did not seem to be physiologically essential in the initiation of the beatings of cardiomyocytes. Potassium currents were partially inhibited by tributyltin (TBT) (1 µM) but not by okadaic acid (two marine pollutants). DNA synthesis was also demonstrated in few cultured cells using BrdU (bromo-2'-deoxyuridine). Observed effects of okadaic acid and TBT demonstrated that cultured heart cells from clam Ruditapes decussatus can be used as an experimental model in marine toxicology.

Rapid protein kinase C-dependent reduction of rat skeletal muscle voltage-gated sodium channels by ciliary neurotrophic factor.

The ciliary neurotrophic factor (CNTF), known to exert long-term myotrophic effects, has not yet been shown to induce a rapid biological response in skeletal muscles. The present in vitro study gives rise to the possibility that CNTF could affect the sodium channel activity implied in the triggering of muscle fibre contraction. Therefore, we investigated the effects of an external CNTF application on macroscopic sodium current (I(Na)) in rat native fast-twitch skeletal muscle (flexor digitorum brevis, FDB) by using a cell-attached patch-clamp technique. The I(Na) peak amplitude measured at a depolarizing pulse from -100 to -10 mV is rapidly reduced in a time- and dose-dependent manner by CNTF (0.01-20 ng ml(-1)). The maximal decrease is 25% after 10 min incubation in 2 ng ml(-1) CNTF. There was no alteration in activation or inactivation kinetics, or in activation curves constructed from current-voltage relationships in the presence of CNTF. In contrast, the relative I(Na) inhibition induced by CNTF is accompanied by a hyperpolarizing shift in the midpoint of the inactivation curves: -6 and -10 mV for the steady-state fast and slow inactivation, respectively. Furthermore, CNTF induces a 5 mV hyperpolarization of the resting membrane potential of the fibres. The effects of CNTF are similar to those of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a protein kinase C (PKC) activator, when no effect is observed in the presence of chelerythrine, a PKC inhibitor. These results suggest that, in skeletal muscle, CNTF can rapidly decrease sodium currents by altering inactivation gating, probably through an intracellular PKC-dependent mechanism that could lead to decreased membrane excitability. The present study contributes to a better understanding of the physiological role of endogenous CNTF.