nystatin-a1 and betadex

We measured the effect of β-cyclodextrin (BCD, a cholesterol scavenger) on water flow across the isolated toad bladder exposed to an osmotic gradient (J(w)) by a gravimetric technique. BCD, when present in the solution bathing the apical side of the bladder, inhibited the increase in J(w) caused by nystatin, a polyene antibiotic that acts by directly binding apical membrane cholesterol. When present in the basolateral bath, BCD inhibited the increase in J(w) caused by basolateral exposure to oxytocin (which binds membrane receptors and stimulates the synthesis of cAMP), but did not alter the response to theophylline (which inhibits hydrolysis of cAMP by cyclic nucleotide phosphodiesterase). The present data are consistent with the notion that agents that increase J(w) by interacting with membrane receptors, which appear to be clustered in cholesterol-rich domains of the basolateral membrane, are altered by cholesterol depletion, whereas agents that do not interact with receptors or other basolateral membrane components are not affected by this treatment. In either case, cholesterol depletion of the apical membrane does not affect the increase in J(w) brought about by an increase in intracellular cAMP concentration.

Topics: Animals; beta-Cyclodextrins; Bufo arenarum; Female; Hypertonic Solutions; Male; Mannitol; Nystatin; Oxytocin; Permeability; Theophylline; Urinary Bladder; Water

CD16b is unique in that it is the only Fc receptor linked to the plasma membrane by a GPI (glycosylphosphatidylinositol) anchor. GPI-anchored proteins often preferentially localize to DRMs (detergent-resistant membranes) that are rich in sphingolipids and cholesterol and play an important role in signal transduction. Even though the responses to CD16b engagement have been intensively investigated, the importance of DRM integrity for CD16b signalling has not been characterized in human neutrophils. We provide direct evidence that CD16b constitutively partitions with both low- and high-density DRMs. Moreover, upon CD16b engagement, a significant increase in the amount of the receptor is observed in high-density DRMs. Similarly to CD16b, CD11b also resides in low- and high-density DRMs. In contrast with CD16b, the partitioning of CD11b in DRMs does not change in response to CD16b engagement. We also provide evidence for the implication of Syk in CD16b signalling and its partitioning to DRMs in resting and activated PMNs (polymorphonuclear neutrophils). Additionally, DRM-disrupting agents, such as nystatin and methyl-beta-cyclodextrin, alter cellular responses to CD16b receptor ligation. Notably, a significant increase in the mobilization of intracellular Ca2+ and in tyrosine phosphorylation of intracellular substrates after CD16b engagement is observed. Altogether, the results of this study provide evidence that high-density DRMs play a role in CD16b signalling in human neutrophils.

Topics: Antigens, CD; beta-Cyclodextrins; Calcium; Cell Membrane; GPI-Linked Proteins; Humans; Macrophage-1 Antigen; Neutrophils; Nystatin; Protein Binding; Receptors, IgG; Signal Transduction

Pseudomonas aeruginosa infection is a serious complication in patients with cystic fibrosis and in immunocompromised individuals. Here we show that P. aeruginosa infection triggers activation of the acid sphingomyelinase and the release of ceramide in sphingolipid-rich rafts. Ceramide reorganizes these rafts into larger signaling platforms that are required to internalize P. aeruginosa, induce apoptosis and regulate the cytokine response in infected cells. Failure to generate ceramide-enriched membrane platforms in infected cells results in an unabated inflammatory response, massive release of interleukin (IL)-1 and septic death of mice. Our findings show that ceramide-enriched membrane platforms are central to the host defense against this potentially lethal pathogen.

Topics: Animals; Apoptosis; beta-Cyclodextrins; Bone Marrow Transplantation; Cells, Cultured; Ceramides; Cyclodextrins; Cystic Fibrosis Transmembrane Conductance Regulator; Enzyme Activation; Epithelial Cells; fas Receptor; Fibroblasts; Filipin; Fluorescent Dyes; Humans; Ionophores; Membrane Microdomains; Mice; Nystatin; Pseudomonas aeruginosa; Pseudomonas Infections; Signal Transduction; Sphingomyelin Phosphodiesterase

This study is focused on the functional significance of neutrophil lactosylceramide (LacCer)-enriched microdomains, which are involved in the initiation of a signal transduction pathway leading to superoxide generation. Treatment of neutrophils with anti-LacCer antibody, T5A7 or Huly-m13, induced superoxide generation from the cells, which was blocked by PP1, a Src kinase inhibitor; wortmannin, a phosphatidylinositol-3 kinase inhibitor; SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor; and H7, an inhibitor for protein kinase C. When promyelocytic leukemia HL-60 cells were differentiated into neutrophilic lineage by dimethyl sulfoxide (DMSO) treatment, they acquired superoxide-generating activity but did not respond to anti-LacCer antibodies. Density gradient centrifugation revealed that LacCer and Lyn were recovered in detergent-insoluble membrane (DIM) of neutrophils and DMSO-treated HL-60 cells. However, immunoprecipitation experiments indicated that LacCer was associated with Lyn in neutrophils but not in DMSO-treated HL-60 cells. Interestingly, T5A7 induced the phosphorylation of Lyn in neutrophils but not in DMSO-treated HL-60 cells. Moreover, T5A7 induced the phosphorylation of p38 MAPK in neutrophils. T5A7-induced Lyn phosphorylation in neutrophil DIM fraction was significantly enhanced by cholesterol depletion or sequestration with methyl-beta-cyclodextrin or nystatin. Collectively, these data suggest that neutrophils are characterized by the presence of cell surface LacCer-enriched glycosphingolipid signaling domain coupled with Lyn and that the ligand binding to LacCer induces the activation of Lyn, which may be suppressibly regulated by cholesterol, leading to superoxide generation through the phosphatidylinositol-3 kinase-, p38 MAPK-, and protein kinase C-dependent signal transduction pathway.

Topics: Antibodies; Antigens, CD; beta-Cyclodextrins; Cell Differentiation; Cell Membrane; Centrifugation, Density Gradient; Cyclodextrins; Dimethyl Sulfoxide; Enzyme Activation; Enzyme Inhibitors; Glycosphingolipids; HL-60 Cells; Humans; Lactosylceramides; Lipids; Mitogen-Activated Protein Kinases; Neutrophils; Nystatin; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Signal Transduction; src-Family Kinases; Superoxides

Binding of either ligand or agonistic antibodies to the death receptor CD95 (APO-1/Fas) induces the formation of the death-inducing signaling complex (DISC). We now show that signal initiation of CD95 in type I cells can be further separated into at least four distinct steps. (i) The first step is ligand-induced formation of CD95 microaggregates at the cell surface. (ii) The second step is recruitment of FADD to form a DISC. This step is dependent on actin filaments. (iii) The third step involves formation of large CD95 surface clusters. This event is positively regulated by DISC-generated caspase 8. (iv) The fourth step is internalization of activated CD95 through an endosomal pathway. The latter step is again dependent on the presence of actin filaments. The data indicate that the signal initiation by CD95 is a complex process actively regulated at various levels, providing a number of new drug targets to specifically modulate CD95 signaling.

Topics: Actin Cytoskeleton; Adaptor Proteins, Signal Transducing; Amino Acid Chloromethyl Ketones; Animals; Anti-Bacterial Agents; Apoptosis; beta-Cyclodextrins; Bridged Bicyclo Compounds, Heterocyclic; Carrier Proteins; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Line; Cell Membrane; Cyclodextrins; Endocytosis; fas Receptor; Fas-Associated Death Domain Protein; Filipin; Fluorescent Dyes; Humans; Ionophores; Ligands; Lymphocytes; Membrane Microdomains; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Models, Biological; Nystatin; Receptor Aggregation; Signal Transduction; Thiazoles; Thiazolidines