Cooperative regulation of extracellular signal-regulated kinase activation and cell shape change by filamin A and beta-arrestins.

beta-Arrestins (betaarr) are multifunctional adaptor proteins that can act as scaffolds for G protein-coupled receptor activation of mitogen-activated protein kinases (MAPK). Here, we identify the actin-binding and scaffolding protein filamin A (FLNA) as a betaarr-binding partner using Son of sevenless recruitment system screening, a classical yeast two-hybrid system, coimmunoprecipitation analyses, and direct binding in vitro. In FLNA, the betaarr-binding site involves tandem repeat 22 in the carboxyl terminus.

Homo- and hetero-oligomerization of beta-arrestins in living cells.

Arrestins are important proteins, which regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous beta-arrestins are believed to function exclusively as monomers, although self-association is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Here the oligomerization status of beta-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged beta-arrestins and resonance energy transfer (BRET and FRET) in living cells.

Ginkgolide B stimulates signaling events in neutrophils and primes defense activities.

Ginkgolide B (GKB) is a bioactive component of the standardized extract from the leaves of the Ginkgo biloba tree (EGb 761), which is used in Chinese and in occidental medicine. GKB is known as a platelet-activating factor receptor antagonist. Here, we provide evidence that GKB per se (0.

The myocardium-protective Gly-49 variant of the beta 1-adrenergic receptor exhibits constitutive activity and increased desensitization and down-regulation.

The beta(1)-adrenergic receptor (beta(1)AR) is a major mediator of catecholamine effects in human heart. Patients with heart failure who were hetero- or homozygous for the Gly-49 variant of the beta(1)AR (Gly-49-beta(1)AR) showed improved long-term survival as compared with those with the Ser-49 genotype. Here, the functional consequences of this polymorphism were studied in cells expressing either variant.

Recruitment of activated G protein-coupled receptors to pre-existing clathrin-coated pits in living cells.

The process of clathrin-mediated endocytosis tightly regulates signaling of the superfamily of seven-transmembrane G protein-coupled receptors (GPCRs). A fundamental question in the cell biology of membrane receptor endocytosis is whether activated receptors can initiate the formation of clathrin-coated pits (CPs) or whether they are simply mobilized to pre-existing CPs. Here, using various approaches, including a dynamic assay to monitor the distribution of CPs and GPCR-beta-arrestin complexes in live HeLa cells, we demonstrate for the first time that activated GPCRs do not initiate the de novo formation of CPs but instead are targeted to pre-existing CPs.

Ornithine alpha-ketoglutarate counteracts the decrease of liver cytochrome P-450 content in burned rats.

The effect of ornithine alpha-ketoglutarate (OKG) on cytochrome P-450 enzyme activities was studied in a well-defined model of injury (burn followed by fasting then subsequent hypocaloric diet) administered to young rats for 3 d. Hepatic microsomes were prepared by ultracentrifugation and levels of cytochromes P-450 were determined spectrophotometrically. The activities of ethoxy-resorufin-O-deethylase (EROD), benzyloxy-resorufin-O-dealkylase (BROD), and erythromycin demethylase were measured as markers of P-450 1A, 2A, and 3A isotypes respectively.

Inflammatory reactions induced by various calcium pyrophosphate crystals.

Various inflammatory reactions can be induced by different crystals responsible for various arthropathies. The aim of this work was to study modifications induced in the rat, locally and at distance, by intrapleural injection of 3 forms of pyrophosphate crystals (CaPP dihydrated monoclinic (M), CaPP dihydrated triclinic (T) and CaPP anhydrous (beta]. The data presented here show that the structure of irritants plays a decisive role in the kinetics of inflammatory reactions from a local and systemic point of view.

Drug activity "in vitro" and "in vivo" on leucocyte chemotaxis.

Drug influences on polymorphonuclear leucocytes (PMN) and particularly on PMN migration was investigated. PMN chemotaxis and random migration were assessed in vitro, after drug incubation in vitro and/or administration in vivo. An inhibiting effect on directed migration induced both by fMLP and C5a was demonstrated with some anti-inflammatory drugs whereas random migration was unaffected.

Reversal effect of isaxonine on modifications of rat polymorphonuclear migration by colchicine.

Using a modified Boyden chamber, random migration and chemotaxis of rat polymorphonuclear leucocytes (PMN) were assessed after incubation in vitro in a solution of colchicine (1.5 X 10(-5) M). The results obtained were compared to random migration or chemotaxis of the same cellular pool treated in vitro or in vivo by N-isopropyl-amino-2-pyrimidine phosphate (isaxonine 4.

In vitro and in vivo effects of piroxicam on rat polymorphonuclear responsiveness after induction of two acute non-specific inflammatory reactions.

The effect of piroxicam on rat polymorphonuclear leucocytes (PMN) has been studied in vitro and in vivo after the induction of two acute, non specific inflammatory reactions (pleurisies induced by calcium pyrophosphate crystals (CaPP) or isologous serum). An inhibition of chemotaxis by piroxicam has been demonstrated by two techniques, the filter and agarose assays in vivo and in vitro. An inhibition of random cell migration has been observed only at the higher drug concentration using agarose assay with CaPP-elicited cells.

Tachykinins: effects on motility and metabolism of rat polymorphonuclear leucocytes.

Chemotactic and chemoluminescent activities of substance P, substance K, kassinin and the substance P fragments SP 4-11, SP 7-11, SP 1-4 have been investigated in order to identify the minimum active molecular structure responsible for rat polymorphonuclear activation. Substance P, SP 4-11 and SP 7-11 stimulated directed locomotion (chemotaxis) and were found to be active also in the chemoluminescence assay while SP 1-4 had no effect. Moreover, all peptides, except substance K and SP 1-4, inhibited the chemotactic response of polymorphonuclears to the peptide formyl-methionyl-leucyl-phenylalanine and, to a minor extent, also to leukotriene B4.

In vitro and in vivo effects of gold salts on chemotaxis and random migration of rat polymorphonuclear leucocytes.

The effect produced by three gold salts (sodium aurothiomalate, allochrysine, auranofin) on chemotaxis and random migration of rat polymorphonuclear leucocytes (PMN) was investigated under various experimental conditions. The drug activity was examined after incubation in vitro or after administration in vivo. PMNs were recruited after the induction of two acute inflammatory reactions (pleurisies induced by isologous serum or a suspension of calcium pyrophosphate (CaPP) crystals).

Effect of some anti-inflammatory drugs on FMLP-induced chemotaxis and random migration of rat polymorphonuclear leucocytes.

The effect of indomethacin, acetyl salicylic acid and niflumic acid on the chemotaxis and random migration of rat polymorphonuclear leucocytes (PMN) was investigated with a modified Boyden chamber technique under various experimental conditions (two cell sources, administration of drugs in vivo or incubation in vitro, modulation of antichemotactic activity of sera obtained from animals with inflammatory reactions). Indomethacin and niflumic acid inhibited the chemotactic responsiveness of cells collected from the rat pleural cavity after induction of two types of acute inflammatory reactions. This action was dose-dependent and appeared after either in vivo administration of the drug or in vitro incubation of the cells with various concentrations of the drug.

Effect of piroxicam on the chemotaxis and random migration of polymorphonuclear leucocytes after induction of immune and non immune inflammations.

The effect of piroxicam on the chemotaxis and random migration of rat polymorphonuclear leucocytes was investigated with two techniques of assessment (filter and agarose assay) under various experimental conditions. The drug was administered in vivo or tested in vitro on polymorphonuclears collected in the pleural cavity after induction of an immune (reverse passive Arthus reaction) or an acute non-specific inflammation (pleurisy induced by a suspension) (1%) of calcium pyrophosphate crystals (CaPP). In all cases a dose-dependent inhibition of chemotaxis was observed, more striking in the case of CaPP elicited cells which were more reactive than cells collected after the induction of the Arthus reaction.

[Effects produced in vitro and in vivo on the migration of rat polymorphonuclear leukocytes by anti-inflammatory drugs used in dentistry].

The effects produced in vivo and in vitro by indometacin, niflumic acid, acetylsalicylic acid and dexamethasone have been studied on rat polymorphonuclear leucocyte migration with the Boyden chamber technique (formyl - methionyl - leucyl - phenylalanine was the chemoattractant). Excepting acetylsalicylic acid, the drugs tested determined an inhibiting effect on leucocyte migration investigated by four experimental approaches. These substances exerted a direct effect and modified the chemotactic reactivity towards the chemotactic peptide after incubation in normal or inflammatory sera.

[Repeated sampling of rat polymorphonuclear leucocytes from the pleural cavity. Application for the study of leucocyte chemotaxis (author's transl)].

A method is described for allowing the sampling of a homogeneous population of polymorphonuclear cells (PMN) from the rat pleural cavity a well as a technique involving the use of a phase-contrast microscope and video-recording system to study the necrotaxis of rat PMN towards a single erythrocyte lysed by ruby-laser irradiation. This allows both the speed of the leucocytes and the degree of their chemotactic response to be determined. The advantages and disadvantages of the experimental technique are discussed along with its application to the study of in vitro and in vivo actions of various drugs on rat PMN chemotaxis.

[Repeated sampling of rat polymorphonuclear leucocytes from the pleural cavity. Application for the study of leucocyte chemotaxis (author's transl)].

A method is described for allowing the sampling of a homogeneous population of polymorphonuclear cells (PMN) from the rat pleural cavity a well as a technique involving the use of a phase-contrast microscope and video-recording system to study the necrotaxis of rat PMN towards a single erythrocyte lysed by ruby-laser irradiation. This allows both the speed of the leucocytes and the degree of their chemotactic response to be determined. The advantages and disadvantages of the experimental technique are discussed along with its application to the study of in vitro and in vivo actions of various drugs on rat PMN chemotaxis.