calcimycin and Nasal-Polyps

The present microelectrode experiments on fused respiratory epithelial cells of cystic fibrosis (CF) origin and non-CF origin aim at characterizing the molecular basis of the Cl- conductances regulated by cyclic adenosine monophosphate (cAMP) or respectively Ca2+, as described in the preceding publication. Cell membrane potential (Vm) and resistance (Rm) were recorded as well as their response to substitution of 90% of bath Cl- by isethionate (delta Vm,ISE), by I- (delta Vm,I), or by other halide anions. Fused CF cells had significantly (P < 0.05) higher control Vm values (-18.0 +/- 9.4 mV, +/- SD, n = 68) than fused non-CF cells (-12.5 +/- 6.6 mV, n = 69) and responded to the Ca2+ ionophore A23187 with an increase in the Vm response to Cl- substitution, but did not respond to forskolin. This indicates that CF cells express only the Ca(2+)-stimulated Cl- conductance. Injection of the antibody M3A7 against a fusion protein containing amino acids 1195 to 1480 of the CF gene product into young, forskolin-stimulated or old non-CF cells decreased delta Vm,ISE and delta Vm,I within 15 min to values observed in CF cells. This indicates inhibition of the cAMP-stimulated Cl- conductance and supports the molecular identity of this conductance with the CF gene product. However, the slow onset of inhibition does not allow secondary effects to be excluded and a slight fall in Rm remains unexplained. Stimulation of the Ca(2+)-regulated Cl- conductance was not impaired. Injection of M3A7 into CF cells or of a control antibody in non-CF cells had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antibodies; Calcimycin; Calcium; Cell Fusion; Cells, Cultured; Chloride Channels; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Conductivity; Epithelium; Humans; Membrane Proteins; Nasal Polyps; Respiratory System

With the aim of further elucidating the role of the epithelial Cl- conductance and its defect in cystic fibrosis (CF) patients we studied the properties and regulation of the Cl- conductance in primary cultures of human nasal polyp epithelia. To facilitate microelectrode punctures and to gain access to the cytoplasmic compartment for injection of antibodies, we prepared giant cells using a polyethylene-glycol fusion technique. The membrane potential (Vm) and resistance (Rm) and their responses to ionic substitutions in the bath were measured under control conditions and in the presence of different secretagogues. In non-CF cells Vm averaged-12.5 mV (SD +/- 6.6 mV, n = 69) and was independent of time after fusion, while Rm dropped from 12.4 +/- 7.3 M omega (n = 51) to 3.5 +/- 5.5 M omega (n = 24) in the 2nd week after fusion. The low Vm values reflected a vanishing K+ conductance in the presence of a dominating Cl- conductance that increased with time. In young cells, a Cl- conductance prevailed which could be stimulated by application of the Ca2+ ionophore, A23187, or of carbachol. As determined in CF cells, it had an outwardly rectifying current/voltage (ilV) relationship and exhibited the selectivity sequence I- > Br- > Cl- > F- > isethionate (ISE-) both in Vm and Rm measurements. With increasing age after fusion, a Cl- conductance prevailed in non-CF cells which could be stimulated by cyclic adenosine monophosphate (cAMP) or forskolin and which was downregulated by A23187. It had a linear ilV relationship and exhibited the selectivity sequence Br- > Cl- > I- > F- > ISE- if determined from Vm measurements, but a sequence of Cl- > Br- > F- = ISE- > I- if determined from Rm measurements. This points to multiple-ion pore behaviour of the respective Cl- channel. In agreement with observations described in the following publication, the results suggest that the cAMP-regulated Cl- conductance corresponds to the CF-gene product while the molecular nature of the Ca(2+)-regulated Cl conductance is not yet known.

Topics: Anions; Calcimycin; Carbachol; Cell Fusion; Cells, Cultured; Chloride Channels; Colforsin; Cyclic AMP; Cystic Fibrosis; Electric Conductivity; Epithelium; Humans; Nasal Polyps; Respiratory System; Signal Transduction

Nasal polyposis is a chronic inflammatory condition of the upper airways characterized by infiltration of activated inflammatory cells, including mast cells, both in the epithelium and in the stroma. The aim of this work was to study human mast cells derived from two different anatomical sites within the same nasal polyp tissue. To this end, we isolated two distinct mast cell populations, one from the epithelial and the other from the stromal layers of individual human nasal polyp tissues. We examined the mediator content of the two mast cell populations and found that stromal mast cells had a significantly higher content of tryptase compared with the epithelial mast cells from the same tissue. In addition, mast cells from the stromal compartment, but not those from the epithelium, released a significant amount of histamine after anti-IgE stimulation. By contrast, both populations released over 50% of the total histamine after non-specific stimuli (A23187 10(-6) M). The content of mediators and the response to immunological activation were not significantly altered in patients receiving topical steroid therapy. It remains to be determined if the observed differences are the result of an intrinsic characteristic of the mast cell populations localized to separate tissue compartments, or reflect a different in vivo exposure to stimuli such as antigens, or different surrounding structural or infiltrating cells. In conclusion, these data provide evidence of functional heterogeneity and differences in mediator content between mast cell subpopulations from a single human tissue. The failure of release of epithelial mast cell mediators from an immunologic stimulus may have implications concerning acute effects of antigen exposure in nasal polyposis.

Topics: Adult; Aged; Aged, 80 and over; Calcimycin; Cell Separation; Edetic Acid; Female; Histamine; Histamine Release; Humans; Immunoglobulin E; Male; Mast Cells; Middle Aged; Nasal Polyps

Platelet-activating factor (PAF) was measured in human nasal polyps obtained from patients with chronic sinusitis. PAF was quantitated by platelet-aggregating activity and was found to be 7.8 +/- 1.8 pg/micrograms phosphorus of polyp phospholipid (mean +/- SE; n = 23). In the polyps, phosphatidylcholine constituted 30% of the total phospholipids, of which 8.5% was the ether-linked type. Incubations of replicates of the fresh polyps in Tyrode's solution in the presence of calcium ionophore A23187 and antihuman immunoglobulin E resulted in 295 and 65% increases in the amount of PAF, respectively. Thus, nasal polyps in humans with chronic sinusitis possess PAF and have the potential to produce PAF upon stimulation. The participation of PAF in nasal polyp formation is discussed.

Topics: Adolescent; Adult; Aged; Antibodies, Anti-Idiotypic; Calcimycin; Child; Chronic Disease; Female; Humans; Immunoglobulin E; Male; Middle Aged; Nasal Polyps; Phospholipids; Platelet Activating Factor; Platelet Aggregation; Sinusitis

Chopped human nasal polyps and bronchial tissue produced lipoxin A4 and isomers of lipoxins A4 and B4, but not lipoxin B4, after incubation with exogenous leukotriene A4. In addition, these tissues transformed arachidonic acid to 15-hydroxyeicosatetraenoic acid. The capacity per gram of tissue to produce lipoxins and 15-hydroxyeicosatetraenoic acid was 3-5-times higher in the nasal polyps. Neither tissue produced detectable levels of lipoxins or leukotrienes after incubation with ionophore A23187 and arachidonic acid. Co-incubation of nasal polyps and polymorphonuclear granulocytes with ionophore A23187 led to the formation of lipoxins, including lipoxins A4 and B4. The results indicate the involvement of an epithelial 15-lipoxygenase in lipoxin formation in human airways.

Topics: Arachidonate 15-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Bronchi; Calcimycin; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotrienes; Lipoxins; Nasal Polyps; Neutrophils

Cystic fibrosis (CF) airway epithelia express a defect in adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of apical membrane Cl- channels. Recent patch-clamp studies have raised the possibility that Ca2+ -dependent mechanisms for the activation of Cl- secretion may be preserved in CF airway epithelia. To determine 1) whether intact normal (N1) and CF airway epithelia exhibit a Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanisms initiate Cl- secretion via activation of an apical membrane Cl- conductance (GCl-), nasal epithelia from N1 and CF subjects were cultured on collagen membranes, and responses to isoproterenol or Ca2- ionophores [A23187 10(-6) M; ionomycin (10(-5)M)] were measured with transepithelial and intracellular techniques. Isoproterenol induced activation of an apical membrane GCl- in N1 cultures but was ineffective in CF. In contrast, in both N1 and CF amiloride-pretreated cultures, A23187 induced an increase in the equivalent short-circuit current that was associated with an activation of an apical membrane Gc1- and was bumetanide inhibitable. A23187 addition during superfusion of the lumen with a low Cl- (3 mM) solution reduced intracellular Cl- activity of CF cells. A Ca2+ ionophore of different selectivity properties, ionomycin, was also an effective Cl- secretagogue in both N1 and CF cultures. We conclude that 1) the A23187 induced Cl- secretion via activation of an apical GCl- in N1 human nasal epithelium, and 2) in contrast to an isoproterenol-dependent path, a Ca2+ -dependent path for GCl- activation is preserved in CF epithelia.

Topics: Adolescent; Adult; Amiloride; Calcimycin; Calcium; Cells, Cultured; Chloride Channels; Chlorides; Cystic Fibrosis; Electric Conductivity; Ethers; Female; Humans; Ion Channels; Ionomycin; Isoproterenol; Male; Membrane Potentials; Membrane Proteins; Nasal Polyps; Reference Values; Turbinates

An airway epithelial cell line (CF/T43) was developed by infecting cultured airway epithelial cells from patients with cystic fibrosis (CF) with the pZIPneoSV(X)1/SV40T retrovirus and selecting for G418 resistance and ion transport properties. The distinctive chloride secretory phenotypes of the CF cell line CF/T43 and a normal cell line (NL/T4) were not perturbed by SV40T-induced cell transformation. Epithelial cell lines generated from CF cells with the SV40T gene can be used to test candidate CF genes and to evaluate the molecular mechanisms responsible for the CF phenotype.

Topics: Amiloride; Antigens, Polyomavirus Transforming; Calcimycin; Cell Line; Cell Membrane; Chloride Channels; Chlorides; Colforsin; Cystic Fibrosis; Electric Conductivity; Epithelium; Ethers; Freeze Fracturing; Humans; Intercellular Junctions; Ion Channels; Ionomycin; Membrane Proteins; Microscopy, Electron; Nasal Polyps; Simian virus 40; Transformation, Genetic

Nasal polyps were obtained from 22 patients undergoing polyp surgery. They were chopped into fragments of approximately 2 mm2, washed free of blood, and passively sensitized with serum from timothy allergen sensitive patients (RAST 30-40%), then, challenged with timothy allergen. Analysis of the incubation medium after a 30 min challenge as assessed by reversed phase high performance liquid chromatography and bioassay revealed the presence of leukotriene (LT) LTB4, LTC4, LTD4 and LTE4 (29 +/- 9, 6.6 +/- 3, 62 +/- 13 & 86 +/- 23 pmol/g tissue wet weight, respectively). The human nasal polyps also released histamine which reached a maximum after approximately 5 min (2.8 +/- 1.28 nmol/g tissue), as measured by radioenzymatic assay. A similar profile of leukotrienes and histamine release was observed when the nasal polyps were stimulated with ionophore A23187. However, the ionophore stimulated release was greater than that observed for the antigen challenge. When human polyps were stimulated with a mixture of the ionophore and arachidonic acid, all the above products as well as 5-hydroxy-eicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE were detected in the medium. These data, taken together, demonstrate that human nasal polyps are able to release significant amounts of leukotrienes and histamine upon both immunological and non-immunological stimulations and that these responses might significantly contribute to the symptoms of allergic rhinitis.

Topics: Allergens; Arachidonic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Histamine Release; Humans; Nasal Polyps; SRS-A; Time Factors