Assessment of chloride secretion in human nasal epithelial cells by X-ray microanalysis

The genetic disease cystic fibrosis (CF) is due to defective epithelial chl oride transport. Different treatments have been proposed that could restore chloride transport in CF patients. A new method is proposed for measuring the chloride secretion in easily accessible epithelial cells. Fresh nasal epithelial cells were obtained by nasal brushing and made to at tach to titanium grids for electron microscopy. Chloride efflux through the cystic fibrosis transmembrane regulator channel was stimulated by 20 mum f orskolin and 100 mum isobutyl-methylxanthine (IBMX), in standard Ringer's s olution (SR). Chloride efflux through the calcium-regulated channel was sti mulated by 200 mum adenosine triphosphate (ATP) in SR. The cells were rinse d after the exposure, in order to remove the experimental medium, frozen an d freeze-dried. The elemental composition of the cells was determined by X- ray, microanalysis. Rinsing with distilled water or ammonium acetate appeared to cause damage t o the cells, whereas rinsing with isotonic mannitol preserved the ionic com position. Stimulation of cells from healthy controls with forskolin and IBM X in a chloride-containing medium caused a significant (28 +/- 6%) decrease in chloride concentration, which is indicative of net chloride efflux. In similar conditions, stimulation with ATP induced a 29 +/- 5% decrease in th e chloride concentration. Stimulation of cells from CF patients with forskolin and IBMX in a chloride -containing medium caused no significant change in the intracellular chlori de concentration, whereas ATP stimulation induced a response similar to tha t obtained in cells from healthy controls. It is concluded that X-ray microanalysis of nasal epithelial cells may be u sed to determine chloride secretion in CF patients in an easily accessible cell type.