The cellular basis of cystic fibrosis (CF) is a defect in a cyclic adenosine monophosphate (cAMP)‐activated chloride channel (CF transmembrane conductance regulator) in epithelial cells that leads to decreased chloride ion transport and impaired water transport across the cell membrane. This study investigated whether it was possible to activate the defective chloride channel in cystic fibrosis respiratory epithelial cells with 4‐phenylbutyrate (4PBA), genistein and 8‐cyclopentyl‐1,3‐dipropylxanthine (CPX). The CF bronchial epithelial cell line CFBE41o‐, which expresses the ΔF508 mutation, was treated with these agents and loss of Cl‐, indicating Cl‐ efflux, measured by X‐ray microanalysis. 8‐bromo‐cAMP alone did not induce Cl‐ efflux in CFBE41o‐ cells, but after incubation with 4PBA a significant efflux of Cl‐ occurred. Stimulation of cells with a combination of genistein and cAMP also induced Cl‐ efflux, whereas a combination of pretreatment with 4PBA and a combined stimulation with genistein and cAMP induced an even larger Cl‐ efflux. Cl‐ efflux could also be stimulated by CPX, but this effect was not enhanced by 4PBA pretreatment. The ΔF508 mutation leads to impaired processing of the cystic fibrosis transmembrane conductance regulator. The increased efflux of chloride after 4‐phenylbutyrate treatment can be explained by the fact that 4‐phenylbutyrate allows the ΔF508 cystic fibrosis transmembrane conductance regulator to escape degradation and to be transported to the cell surface. Genistein and 8‐cyclopentyl‐1,3‐dipropylxanthine act by stimulating chloride ion efflux by increasing the probability of the cystic fibrosis transmembrane conductance regulator being open. The combination of 4‐phenylbutyrate and genistein may be useful in a potential pharmacological therapy for cystic fibrosis patients with the ΔF508 mutation.

Effects of TNF-a, IFN-c and IL-b on normal human bronchial epithelial cells. C. Kampf, A.J. Relova, S. Sandler, G.M. Roomans. #ERS Journals 1999. ABSTRACT: Several diseases affecting the airways such as asthma are associated with both epithelial damage and increased levels of pro-inflammatory cytokines. To investigate the possible relation between cytokines and epithelial damage, the effects of tumour necrosis factor-a (TNF)-a, interferon gamma (IFN-c) and interleukin-1 beta (IL-1b) on normal human bronchial epithelial cells in vitro were studied.The cells were exposed to these cytokines for 48 or 72 h, followed by morphological, immunohistochemical and metabolic studies.Transmission and scanning electron microscopical analyses demonstrated damage to the mitochondria and an increase in cell processes induced by the cytokines. The use of antibodies against desmosomal cytokeratin showed a decrease in desmosome formation in IFN-c-exposed cells. Decreased glucose oxidation rate and increased accumulation of nitric oxide were found in cytokine-exposed cells. N v -monomethyl-Larginine (L-NMMA) reduced nitrite production. X-ray microanalysis showed an increase in the intracellular sodium/potassium ratio of the cells after exposure to cytokines, which is an indication of cell damage. The cytokines induced both necrosis and apoptosis to varying degrees. IFN-c and TNF-a generally potentiate each other's effects.In conclusion tumour necrosis factor-a and interferon gamma, and to a lesser extent interleukin-1b, can cause damage to epithelial cells, which may be a factor involved in epithelial shedding in airway diseases.

Nasal polyposis is a poorly understood chronic inflammatory disease often associated with asthma. As nasal polyps and asthma both are associated with massive eosinophil infiltration, they may share a common pathophysiological mechanism. Many genetic and autoimmune diseases may result from altered expression or function of cell adhesion molecules such as desmosomes. A transmission electron microscopical study was carried out on tissue from 15 patients suffering from nasal polyps, to investigate if there are changes in desmosomes in nasal polyps from asthmatic and/or allergic patients versus non-asthmatic versus non-allergic patients. In allergic patients the damage to columnar cells was more extensive than in non-allergic patients. Massive infiltration of eosinophils was observed in epithelium and connective tissue in all groups. No significant difference in thickness of the basal lamina was found between any of the groups. All patients had dilated capillaries in the connective tissue. The intercellular space between the epithelial cells was smallest in the asthmatic non-allergic group. The relative length of columnar cell or basal cell desmosomes was reduced in patients with asthma or allergy, compared to non-allergic, non-asthmatic patients. Hence, there appears to be a weakness in the desmosomes in asthmatics and allergics. Epithelial shedding may play an important role in the pathophysiological process of a multifactorial disease such as asthma.

Electron probe X-ray microanalysis has been used for the last 25 years by biologists to obtain information about the distribution of elements at the cell and tissue level. During this period, progress has mainly been made through the development of more adequate techniques for specimen preparation (mainly low temperature techniques) and quantitative analysis, so that accurate analysis of the physiologically important cellular ions can be carried out. Use of in vitro systems and cell cultures may further increase the number of problems to which X-ray microanalysis can be applied. Among the numerous applications of X-ray microanalysis in cell biology and cell pathology, applications in the areas of epithelial ion transport, the role of calcium in secretory and contractile cells, and the role of ions in cell proliferation and cancer are discussed in more detail.

cAMP-induced ion transport in a human sweat gland cell line, NCL-SG3, was investigated by X-ray microanalysis and patch-clamp technique. Stimulation with cAMP caused a decrease in the cellular Cl and K. cAMP had no significant effect on the intracellular Na and Ca. Chloride channel blockers (9-AC, DPC and NPPB) inhibited the cAMP-induced chloride efflux. In patch-clamp experiments the inward current increased over a period of 5-15 min on addition of membrane-permeable cAMP in 66% of the attempts when the cell was held at 0 mV and pulsed to negative membrane potentials. The inward current was completely blocked by chloride channel blockers. Washout reversed the effect of cAMP. The inward current was not diminished by substitution of impermeable cations for Na in the bath and was insensitive to TEA (tetraethylammoniumchloride). It is concluded that the inward current is mainly a chloride current. Cystic fibrosis transmembrane regulator (CFTR) could not be demonstrated in the NCL-SG3 cells. It is therefore possible that the chloride efflux is mediated by other types of chloride channels.