[Culture conditions for gametes and embryos: Which culture medium? Which impact on newborn?] / Conditions de culture pour les gamètes et embryons : quels milieux de culture ? et quelle incidence sur le nouveau-né ?

Many studies have examined the impact of cell/embryo culture media on the development of human embryo during IVF process, but few studies have followed up and compared the effects of these culture media on the developmental outcome of children conceived by IVF. As recurrent experimental evidence from animal studies suggests potential long-term effects of embryo culture media on the health outcome of IVF-conceived children, more studies are needed to clarify the role of the culture media and mechanisms underlying such effects. In human, however, the effects of culture media are difficult to pinpoint due to complications stem from both the influence of maternal nutrition during the gestational period and the parental genetic. Based on a simple review of the literature integrating animal experimentations and human clinic studies, we suggest that the composition of culture medium should be considered beyond the character of unique or sequential medium, corresponding to "let embryo choose" or "back to nature" respectively. Instead, we suggest that the main components of embryo culture media should be considered from the point of view of metabolic consequences and potential epigenetic effects. Given that energetic metabolites can regulate epigenetic machinery, we hypothesize that metabolic abnormalities linked to morphological abnormalities could reveal epigenetic defects in embryos.

[Extracorporeal CO2 Elimination (ECCO2R) for Hypercapnic Respiratory Failure: From Pathophysiology to Clinical Application]. / Extrakorporale CO2-Elimination (ECCO2R): von der Pathophysiologie zur klinischen Anwendung beim hyperkapnischen respiratorischen Versagen.

Extracorporeal CO2 removal (ECCO2R) is becoming an increasingly established treatment option for patients with acute severe hypercapnic respiratory failure. Technically, pumpless arterio-venous systems using the natural arterio-venous pressure gradient and also pump-driven veno-venous systems are available. Here, veno-venous ECCO2R has become the preferred technique, as settings for arterio-venous ECCO2R are restricted and side effects are more common with arterio-venous ECCO2R. Using veno-venous ECCO2R with blood flow rates up to 450 ml/min 60 to 80 ml CO2 can be removed per minute corresponding to 20 to 30 % of the total amount of CO2 production. However, in case of very severe hypercapnic respiratory failure with severe respiratory acidosis (pH 7.1 or less) blood flow rates of around 1000 ml/min are required for compensating severe respiratory acidosis corresponding to the elimination of 50 to 60 % of the total amount of CO2 production. Relevant side effects include the activation of blood coagulation and associated bleeding complications. Two recent case-control studies in severely exacerbated COPD patients could demonstrate that intubation rates can be reduced by the application of ECCO2R, but this was associated with non-ignorable side effects. Therefore, randomized controlled trials are urgently needed to more precisely establish the risks and benefits of ECCO2R when aimed at avoiding intubation.

[Role of the vascular wall in the pathophysiology of the acute coronary syndrome]. / Die Rolle der Gefässwand in der Pathophysiologie des akuten Koronarsyndroms.

The endothelium is of important significance in the development of the acute coronary syndrome. As an endo-/paracrine organ, the endothelium plays a key role in the regulation of the vascular homeostasis. The endothelial integrity and above all the bioavailability of nitric oxide (NO) are essential for the correct function of the endothelium. Cardiac risk factors may lead to an endothelial dysfunction with a consecutive imbalance of the vascular homeostasis. In an inflammatory or prothrombotic state the endothelium shows a number of abnormalities such as oxidative stress, expression of cell adhesion molecules, activation of cell signal-systems (renin-angiotensin-system, CD40/CD40L-system) and especially the loss of NO. The inflammatory cascades lead to coronary atherosclerosis over years or, more instantly, to the acute coronary syndrome caused by endothelial erosion or the rupture of an instable plaque. The knowledge of the pathophysiological processes in the arterial wall during the acute coronary syndrome may lead to the identification of high risk patients and the development of more targeted therapies.

Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells.

The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na(+) absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na(+), Mg(2+), P, S and Cl(-)) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR(inh)-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.

Defective remethylation of homocysteine is related to decreased synthesis of coenzymes B2 in thyroidectomized rats.

We investigated the influence of hypothyroidism on homocysteine metabolism in rats, focusing on a hypothetical deficient synthesis of FAD by riboflavin kinases. Animals were allocated in control group (n = 7), thyroidectomized rats (n = 6), rats with diet deficient in vitamin B2, B9, B12, choline and methionine (n = 7), thyroidectomized rats with deficient diet (n = 9). Homocysteine was decreased in operated rats (2.6 +/- 1.01 vs. 4.05 +/- 1.0 mumol/L, P = 0.02) and increased in deficient diet rats (29.56 +/- 4.52 vs. 4.05 +/- 1.0 micromol/L, P = 0.001), when compared to control group. Erythrocyte-Glutathione-Reductase-Activation-Coefficient (index of FAD deficiency) was increased in thyroidectomized or deficient diet rats (P = 0.004 for both). Methylenetetrahydrofolate-reductase and methionine-synthase activities were decreased in thyroidectomized rats but not in those subjected to deficient diet. Cystathionine-beta-synthase was increased only in operated rats. Taken together, these results showed a defective re-methylation in surgical hypothyroidism, which was due in part to a defective synthesis of vitamin B2 coenzymes. This defective pathway was overcompensated by the increased Cystathionine-beta-synthase activity.

P-selectin in arterial thrombosis.

P-selectin is a transmembrane protein present in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells. Following activation, it is rapidly translocated to the cell surface. P-selectin expression in platelets has been shown to be elevated in disorders associated with arterial thrombosis such as coronary artery disease, acute myocardial infarction, stroke, and peripheral artery disease. P-selectin mediates rolling of platelets and leukocytes on activated endothelial cells as well as interactions of platelets with leukocytes. Platelet P-selectin interacts with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes to form platelet-leukocyte aggregates. Furthermore, this interaction of P-selectin with PSGL-1 induces the upregulation of tissue factor, several cytokines in leukocytes and the production of procoagulant microparticles, thereby contributing to a prothrombotic state. P-selectin is also involved in platelet-platelet interactions, i. e. platelet aggregation which is a major factor in arterial thrombosis. P-selectin interacts with platelet sulfatides, thereby stabilizing initial platelet aggregates formed by GPIIb/IIIa-fibrinogen bridges. Inhibtion of the P-selectin-sulfatide interaction leads to a reversal of platelet aggregation. Thus, P-selectin plays a significant role in platelet aggregation and platelet- leukocyte interactions, both important mechanisms in the development of arterial thrombosis.

Sulfatides: targets for anti-phospholipid antibodies.

BACKGROUND: Sulfatides are sulfated glycosphingolipids expressed on the surface of erythrocytes, leukocytes, and platelets. Sulfatides interact with several cell adhesion molecules involved in hemostasis. Beta2-glycoprotein I is an anionic phospholipid-binding plasma protein, and the phospholipid-bound form is the target for most anti-phospholipid antibodies that are associated with recurrent thrombosis, miscarriages, and neurological symptoms. In this study, we examined whether beta2-glycoprotein I forms a complex with sulfatides and thereby becomes a target for anti-phospholipid antibodies. METHODS AND RESULTS: Beta2-glycoprotein I binds to surface-bound sulfatides but not to other glycolipids, such as ceramide, cerebrosides, sphingomyelin, or ganglioside. At a sulfatide coating density of 1 microg/well, beta2-glycoprotein I reaches half-maximal binding at 2.5 microg/mL, and the binding is saturated at 10 microg/mL. The binding of beta2-glycoprotein I also depends on the coating density of sulfatides in the well. At a constant beta2-glycoprotein I concentration of 5 microg/mL, maximal binding of beta2-glycoprotein I is observed at a coating density of 1 mug/well. The serum from 14 patients with anti-cardiolipin antibodies, a subset of anti-phospholipid antibodies, bound to sulfatide-bound beta2-glycoprotein I and previous absorption on cardiolipin-coated surfaces decreased the immunoreactivity toward sulfatide-beta2-glycoprotein I complex by >50% in 12 of 14 patients. Furthermore, immunoaffinity-purified anti-cardiolipin antibodies from 4 of 5 patients reacted with sulfatide-bound beta2-glycoprotein I. CONCLUSIONS: These results show that not only anionic phospholipids, as commonly known, but also sulfatides are targets for most anti-phospholipid antibodies. We therefore postulate that interactions of these antibodies with sulfatides may contribute to some of the clinical symptoms of the anti-phospholipid antibody syndrome.

Role for sulfatides in platelet aggregation.

BACKGROUND: Sulfatides are sulfated glycosphingolipids present on the surface of oligodendrocytes, renal tubular cells, and certain tumor cells. They appear to be involved in nerve conduction and cell adhesion, but their precise physiological function is not known. METHODS AND RESULTS: Here, we show a novel role for sulfatides as a major ligand for P-selectin in platelet adhesion and aggregation. Sulfatides are expressed on the platelet surface, and platelets expressing sulfatides adhere to P-selectin. Both sulfatide micelles and sulfatide-binding recombinant malaria circumsporozoite protein (MCSP) inhibit this adhesion. In parallel, platelets and CHO cells expressing P-selectin adhere to sulfatides, and anti-P-selectin antibodies inhibit this adhesion. Furthermore, both anti-P-selectin antibodies and sulfatide antagonist MCSP significantly reverse platelet aggregation induced by ADP, collagen, or thrombin receptor-activating peptide, suggesting that sulfatide-P-selectin interactions are necessary for the formation of stable platelet aggregates. CONCLUSIONS: These results show that sulfatide interactions with P-selectin are important in platelet adhesion and platelet aggregation. The sulfatide interactions with P-selectin stabilize platelet aggregates, representing a new mechanism of platelet aggregation that may play a significant role in hemostasis and thrombosis.

Influence of culture conditions on the alpha 1,2-fucosyltransferase and MUC gene expression of a transformed cell line MM-39 derived from human tracheal gland cells.

Human tracheal glands cells (HTGC) in culture are able to respond to adrenergic, cholinergic and purinergic agonists by increasing their serous and mucin secretions. These secretagogues are also able to maintain an optimal responsiveness of serous cells to stimulation when they are regularly and briefly delivered to the cells, making the HTGC a suitable model to study the serous secretion (Merten, in press). Our interest has been focused on the effects of cholinergic and purinergic secretagogues associated to histamine, on the mucous function of the transformed human tracheal gland cell line MM-39, which has a mixed, both serous and mucous, phenotype. When the cells were exposed to short stimulation every 2 days for 3 weeks with 10 or 100 microM carbachol, UTP and histamine, modifications of their mucous phenotype were observed. The expression of MUC genes appeared dependent on the culture conditions. Transcripts of MUC1, MUC4, and MUC5B genes were observed when the cells were regularly exposed to the mixture of secretagogues at a concentration of 10 microM, in contrast to the unstimulated expression of MUC1 and MUC4 in control cells. MUC1, MUC4, MUC7, MUC6 and MUC11 transcripts were observed when the cells were regularly exposed to the mixture of secretagogues at a concentration of 100 microM. These culture conditions were also able to induce an alpha 1,2-fucosyltransferase activity absent in the MM-39 cells cultivated with standard conditions. There was no marked effect on the alpha 2,3-sialyltransferase activity although the expression pattern of the sialyltransferase genes was reduced to the unique presence of ST3Gal III. In conclusion, MM-39 cells exposed to repeated stimulation by secretagogues at different concentrations express different sero-mucous phenotypes.

Cholesterol sulfate: a new adhesive molecule for platelets.

BACKGROUND: Cholesterol 3-sulfate is present on a variety of cells and in human LDL, and it has been found in atherosclerotic lesions of human aorta. Its precise biological role has not yet been described. METHODS AND RESULTS: In this study, we investigated the interaction of platelets with cholesterol sulfate. Platelets adhered in a concentration-dependent and saturable manner to cholesterol sulfate but did not adhere to cholesterol, cholesterol acetate, estrone sulfate, or dehydroepiandrosterone sulfate, suggesting that the specificity of this interaction is determined not only by the cholesterol moiety but also by the sulfate group. This adhesion did not increase after platelet activation, and it was not cation-dependent. Soluble cholesterol sulfate inhibited adhesion in a concentration-dependent manner. However, antibodies against glycoprotein Ib, glycoprotein IIb/IIIa, CD36, P-selectin, von Willebrand factor, or thrombospondin had no significant effect on platelet adhesion to cholesterol sulfate. Perfusion of whole blood in a parallel-plate flow chamber resulted in the rapid and progressive adhesion of platelets to cholesterol sulfate but not to cholesterol acetate or estrone sulfate. CONCLUSIONS: Cholesterol sulfate supports platelet adhesion and may be one of the factors determining the prothrombotic potential of atherosclerotic lesions.

Influence of TNFalpha on the sialylation of mucins produced by a transformed cell line MM-39 derived from human tracheal gland cells.

In order to investigate the influence of inflammation on the peripheral glycosylation of airway mucins, a human respiratory glandular cell line (MM-39) was treated by TNFalpha. The expression and the activity of sialyl- and fucosyl-transferases, involved in the biosynthesis of peripheral carbohydrate determinants like sialyl-Lewis x, were investigated by RT-PCR and by HPAEC respectively. The mRNA steady-state level of sialyl- (ST3Gal III) and of fucosyl- (FUT3) transferases was moderately up-regulated by TNFalpha; a 52% increase of alpha2,3-sialyltransferase activity was also observed in TNFalpha-stimulated MM-39 cells. After metabolic radio-labelling with [(3)H]glucosamine and [(3)H]fucose, the mucins released in the culture supernatant were purified by Sepharose CL-4B, density-gradient centrifugation and treatment with glycosaminoglycans-degrading enzymes. The mucins, released in the culture supernatant from control MM-39 cells, were constituted by two populations of molecules having the same 1.39-1.44 mg/ml density but carrying either high or low amounts of sialic acid residues at their periphery. TNFalpha was able to increase the sialylation of the weakly sialylated mucins. This effect and the enhancement of the alpha2,3-sialyltransferase activity by TNFalpha argue in favour of a regulation of the mucin sialylation by this pro-inflammatory cytokine. Despite the moderate overexpression of FUT3, no fucosylation of mucins produced by MM-39 cells was induced by TNFalpha. In conclusion, the influence of TNFalpha on the sialylation of mucins could explain why the mucins from infected patients suffering either from cystic fibrosis or from chronic bronchitis are more sialylated.

P-selectin expression on platelets determines size and stability of platelet aggregates.

BACKGROUND: P-selectin mediates rolling of platelets and leukocytes on activated endothelial cells. After platelet activation, P-selectin is translocated from intracellular granules to the external membrane, whereas fibrinogen aggregates platelets by bridging glycoprotein (GP) IIb/IIIa between adjacent platelets. METHODS AND RESULTS: In this study, we define a novel role for P-selectin in platelet aggregation. Expression of P-selectin on the platelet surface correlated strongly with the mean platelet aggregate size. Inhibition of P-selectin binding to its ligand by either monoclonal anti-P-selectin antibodies directed against the lectin domain or soluble human P-selectin reversed platelet aggregation even when added up to 5 minutes after activation; however, fibrinogen binding to platelets was not affected. This deaggregating effect significantly reduced the maximal size and number of platelet aggregates. When added 1 minute after platelet activation, anti-P-selectin antibody achieved 95% to 100% of the deaggregating effect of EDTA, whereas the anti-GP IIb/IIIa antibody abciximab had no effect. Monoclonal antibodies against known P-selectin ligands, such as P-selectin GP ligand-1 (PSGL-1) or GP Ib, had no effect on platelet aggregation, suggesting a different ligand for P-selectin in platelet aggregate stabilization. In kinetic studies, P-selectin was maximally expressed 10 minutes after platelet activation, whereas maximal activation of GP IIb/IIIa occurred within the first 10 seconds, suggesting that P-selectin operates after fibrinogen binding to activated GP IIb/IIIa. CONCLUSIONS: These results indicate that P-selectin interaction with a ligand, different from PSGL-1 or GP Ib, stabilizes initial GP IIb/IIIa-fibrinogen interactions, allowing the formation of large stable platelet aggregates.

A new role for P-selectin in shear-induced platelet aggregation.

BACKGROUND: P-selectin, expressed on platelets on activation, mediates rolling of platelets on endothelial cells, but its role in shear-induced platelet aggregation is not known. METHODS AND RESULTS: Platelets were exposed to either a single pulse (30 seconds) or 3 pulses (10 seconds) of high shear stress (150 to 200 dynes/cm(2)) each followed by low shear stress (10 dynes/cm(2)) for 4.5 minutes or 90 seconds, respectively, at 37 degrees C to resemble more closely in vivo conditions such as those in stenotic arteries. Under these conditions, platelet aggregation was significantly increased compared with low or high shear stress alone. Monoclonal anti-P-selectin antibodies inhibited shear-induced platelet aggregation, especially when induced by the combination of high and low shear stress, by approximately 70% and had an additive effect on the inhibition by abciximab (anti-glycoprotein (GP) IIb/IIIa antibody). However, anti-P-selectin antibody inhibited shear-induced platelet aggregation only at 37 degrees C, not at 22 degrees C, whereas abciximab inhibited shear-induced platelet aggregation at both 22 degrees C and 37 degrees C. This differential effect of anti-P-selectin antibody is explained by the finding that shear-induced P-selectin expression on platelets was observed mainly at 37 degrees C. CONCLUSIONS: These results indicate that pulsatile shear stress, which resembles flow conditions in stenotic arteries, induces significantly more platelet aggregation at 37 degrees C than monophasic shear stress. Under these conditions, we show a novel role for P-selectin in platelet aggregation distinct from that of GP IIb/IIIa, which may be of importance in the initiation of thrombosis associated with atherosclerotic lesions.

Histidylated polylysine as a synthetic vector for gene transfer into immortalized cystic fibrosis airway surface and airway gland serous cells.

BACKGROUND: We recently designed a cationic polymer called histidylated polylysine made of polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated polylysine complexes. METHODS: and results Here, we have analyzed the ability of histidylated polylysine to transfer reporter or CFTR genes into immortalized cystic fibrosis airway surface epithelial cells (sigmaCFTE29o- cells) and airway gland serous cells (CF-KM4 cells) which are both important targets for cystic fibrosis gene therapy. The luciferase reporter gene expression measured after gene transfer with histidylated polylysine into both cell lines was quite high and similar to that obtained with commercially available vectors. In addition, the level of expression was not dependent on the presence of a membrane disrupting agent such as chloroquine. Histidylated complexes were present in slightly acidic non-lysosomal cellular compartments as shown by a cytological approach using biotinylated plasmid, lysosome-specific antibodies and confocal microscopy. Histidylated complexes appeared to be of small size when prepared at low ionic strength and formed aggregates upon increasing the ionic strength. However, aggregate formation was prevented by the addition of 10% fetal bovine serum. Gene transfer efficiency varied with the size of the complexes and decreased when small particles were used. CONCLUSIONS: These results suggest that histidylated polylysine may be an efficient non-viral vector for gene transfer into cystic fibrosis airway surface epithelial cells and airway gland serous cells.

A quantitative multistandard reverse transcriptase-polymerase chain reaction assay of the cystic fibrosis transmembrane conductance regulator: its usefulness in studying efficiency of gene transfer.

Procedures to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels have already been described but are not universally accepted, and many investigators are skeptical about quantification. To be able to accurately monitor gene therapy, we developed a quantitative multistandard RT-PCR method. This was based on the observation that the CFTR and ribosomal phosphoprotein PO (PR-PO) genes have retained important sequence homologies between rat and human species, allowing the use of rat RNA as an internal standard. A mixture of rat and human RNAs is simultaneously reverse-transcribed in one reaction tube and amplification of CFTR leads to rat and human amplificates with identical sizes which will be discriminated by restriction analysis. PR-PO is analyzed similarly and serves as a control of template loading. RT-PCR of different amounts of RNAs gave similar CFTR/PR-PO ratios, with a coefficient variation below 10%. This technique was applied to a cell line of cystic fibrosis tracheal gland serous cells (CF-KM4) incubated with a recombinant adenovirus containing the CFTR cDNA. Kinetics and dose dependency of transgene expression could be accurately quantified. This method is precise, reproducible, and very simple and could be applied to monitor gene therapy in minute amounts of tissue such as biopsies from cystic fibrosis patients.

Efficient gene transfer into human normal and cystic fibrosis tracheal gland serous cells with synthetic vectors.

Submucosal gland serous cells are believed to play a major role in the physiopathology of cystic fibrosis (CF) and may represent an important target for CF gene therapy. We have studied the efficiency of reporter gene transfer into immortalized normal (MM-39) and CF (CF-KM4) human airway epithelial gland serous cells using various synthetic vectors: glycosylated polylysines (glycofectins), polyethylenimine (PEI) (25 and 800 kD), lipofectin, and lipofectAMINE. In both cell lines, a high luciferase activity was achieved with various glycofectins, with PEI 25 kD, and with lipofectAMINE. After three transfections applied daily using alpha-glycosylated polylysine, 20% of the cells were transfected. At 24 h after CF transmembrane conductance regulator (CFTR) gene transfer into CF-KM4 cells using alpha-glycosylated polylysine, the immunolocalization of CFTR was analyzed by laser scanning confocal microscopy and the transgenic CFTR was detected by an intense labeling of the plasma membrane. The presence of membrane lectins, i. e., cell surface receptors binding oligosaccharides, was also examined on MM-39 and CF-KM4 cells by assessing the binding and uptake of fluorescein-labeled neoglycoproteins and fluorescein-labeled glycoplexes (glycofectins complexed to plasmid DNA). Among all the neoglycoproteins and glycoplexes tested, those bearing alpha-mannosylated derivatives were most efficiently taken up by both normal and CF gland serous cells. However, alpha-mannosylated polylysine was quite inefficient for gene transfer, indicating that the efficiency of gene transfer is determined both by the uptake of the complexes and also by their intracellular trafficking. Moreover, our results show that an efficient in vitro gene transfer was achieved in human airway gland serous cells with the same synthetic vectors described to efficiently transfect human airway surface epithelial cells.

Characterization of a diadenosine tetraphosphate-receptor distinct from the ATP-purinoceptor in human tracheal gland cells.

Human submucosal tracheal glands are now believed to play a major role in the physiopathology of cystic fibrosis, a genetic disease in which ATP is used as a therapeutic agent. However, actions of ATP on tracheal gland cells are not well known. ATP binds to P2 receptors and induced secretory leucocyte protease inhibitor (SLPI) secretion through formation of cyclic adenosine monophosphate and mobilization of intracellular [Ca(2+)]. Since diadenosine polyphosphates (ApnA) are also endogenous effectors of P2 receptors, we investigated their effects in a cell line (MM39) of human tracheal gland cells. Diadenosine tetraphosphates (Ap4A) induced significant stimulation (+50+/-12%) of SLPI secretion and to a similar extent to that of ATP (+65+/-10%). No significant effects were observed with diadenosine triphosphate (Ap3A), diadenosine pentaphosphate (Ap5A), ADP and 2-methylthio-adenosine triphosphate (2-MeS-ATP). Since Ap4A was weakly hydrolyzed (<2% of total), and the hydrolysis product was only inosine which is ineffective on cells, this Ap4A effect was not due to Ap4A hydrolysis in ATP and adenosine monophosphate (AMP). A mixture of Ap4A and ATP elicited only partial additive effects on SLPI secretion. ADP was shown to be a potent antagonist of ATP and Ap4A receptors, with IC(50)s of 0.8 and 2 microM, respectively. 2-MeS-ATP also showed antagonistic properties with IC(50)s of 20 and 30 microM for ATP- and Ap4A-receptors, respectively. Single cell intracellular calcium ([Ca(2+)](i)) measurements showed similar transient increases of [Ca(2+)](i) after ATP or Ap4A challenges. ATP desensitized the cell [Ca(2+)](i) responses to ATP and Ap4A, and Ap4A also desensitized the cell response to Ap4A. Nevertheless, Ap4A did not desensitize the cell [Ca(2+)](i) responses to ATP. In conclusion, both P2Y2-ATP-receptors and Ap4A-P2D-receptors seem to be present in tracheal gland cells. Ap4A may only bind to P2D-receptors whilst ATP may bind to both Ap4A- and ATP-receptors.

Development of substituted Benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel.

Chloride channels play an important role in the physiology and pathophysiology of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), which we show to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We examined the effect of MPB compounds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovary (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Single and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reveal that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native respiratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MPB compounds was glibenclamide-sensitive and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, MPB drugs activate CFTR channels and stimulate the secretion of the antibacterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secretion without changing intracellular cAMP and ATP levels. Similarly, in CHO cells MPB compounds have no effect on the intracellular levels of cAMP and ATP or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or alkaline phosphatase). Our results provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion and therefore represent a new tool to study CFTR-mediated chloride and secretory functions in epithelial tissues.

Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone inhibits expression of P2Y receptors in cystic fibrosis tracheal gland cells.

ATP and UTP have been proposed for use as therapeutic treatment of the abnormal ion transport in the airway epithelium in cystic fibrosis (CF), the most characteristic feature of which is permanent infection by Pseudomonas aeruginosa. As for diverse gram-negative bacteria, this pathogenic bacterium accumulates diffusible N-acylhomoserine lactone (AHL) signal molecules, and when a threshold concentration is reached, virulence factor genes are activated. Human submucosal tracheal gland serous (HTGS) cells are believed to play a major role in the physiopathology of CF. Since ATP and UTP stimulate CF epithelial cells through P2Y receptors, we sought to determine whether CF HTGS cells are capable of responding to the AHLs N-butanoyl-L-homoserine lactone (BHL), N-hexanoyl-L-homoserine lactone (HHL), N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), with special reference to P2Y receptors. All AHLs inhibited ATP- and UTP-induced secretion by CF HTGS cells. The 50% inhibitory concentrations were as high as 10 and 5 microM for BHL and HHL, respectively, but were only 0.3 and 0.4 pM for OdDHL and OHHL, respectively. Furthermore, all AHLs down-regulated the expression of the P2Y2 and P2Y4 receptors. Ibuprofen and nordihydroguaiaretic acid were able to prevent AHL inhibition of the responses to nucleotides, but neither dexamethasone nor indomethacin was able to do this. These data indicate that AHLs may alter responsiveness to ATP and UTP by CF HTGS cells and suggest that, in addition to ATP and/or UTP analogues, ibuprofen may be of use for a combinational pharmacological therapy for CF.

Platelet microparticles promote platelet interaction with subendothelial matrix in a glycoprotein IIb/IIIa-dependent mechanism.

BACKGROUND: Platelets, on activation, release vesicular particles called platelet microparticles. Despite their procoagulant activity, their functional role in platelet-vessel wall interactions is not known. METHODS AND RESULTS: We examined the binding of microparticles to vessel wall components in vitro and in vivo. Microparticles bound to fibrinogen-, fibronectin-, and collagen-coated surfaces. Compared with activated platelets, we observed minimal binding of microparticles to vitronectin and von Willebrand factor. The glycoprotein IIb/IIIa (GP IIb/IIIa) inhibitors abciximab and eptifibatide (Integrilin) inhibited the binding to fibrinogen and fibronectin but had minimal effect on binding to collagen. Furthermore, monoclonal antibodies to GP Ib or anionic phospholipid-binding proteins (beta2-glycoprotein I or annexin V) had no effect in these interactions. Microparticles did not bind to monolayers of resting or stimulated human umbilical vein endothelial cells (HUVECs), even in the presence of fibrinogen or von Willebrand factor. However, under similar conditions, microparticles bound to extracellular matrix produced by cultured HUVECs. Abciximab inhibited this interaction by approximately 50%. In a rabbit model of arterial endothelial injury, the infusion of 51Cr-labeled microparticles resulted in a 3- to 5-fold increase of microparticle adhesion to the injured site compared with the uninjured site (P<0.05%). Furthermore, activated platelets bound to surface-immobilized microparticles in a GP IIb/IIIa-dependent mechanism. This binding increased in the presence of fibrinogen by approximately 30%. CONCLUSIONS: Platelet microparticles bind to subendothelial matrix in vitro and in vivo and can act as a substrate for further platelet binding. This interaction may play a significant role in platelet adhesion to the site of endothelial injury.