Osmotic water permeability (P(f )) was measured in spheroid-shaped human nasal airway epithelial explants pre-exposed to increasing levels of hyperosmotic stress. The fluid-filled spheroids, derived from nasal polyps, were lined by a single cell layer with the ciliated apical cell membrane facing the outside. The P(f ) was determined from diameter changes of the spheroids in response to changes in bathing medium osmolarity forth and back between 300 and 225 mOsm x l(-1). Continuous diameter measurements also allowed determination of spontaneous fluid absorption. Hyperosmotic pretreatment (increase from 300 up to 600 mOsm x l(-1)) caused a time- and osmolarity-dependent increase (up to approximately 1.5 times) in epithelial P(f ) which was of similar magnitude in cystic fibrosis (CF) and non-CF spheroids. The effect saturated at approximately 450 mOsm x l(-1) and at approximately 24 h. Expression of aquaporin-5 (AQP5), studied by immunofluorescence and confocal microscopy, showed an increase in parallel with the increase in P(f ) following hyperosmotic stress. The AQP5 was localized both in cytoplasmic vesicles and in apical cell membranes. Spontaneous fluid absorption rates were equal in CF and non-CF spheroids and were not significantly influenced by hyperosmotic stress. The results suggest that hyperosmotic stress is an important activator of AQP-5 in human airway epithelium, leading to significantly increased transepithelial water permeability.

In order to examine whether calcium-dependent binding of annexin to acidic phospholipids could change the lipid bilayer environment sufficiently to perturb channel-mediated transmembrane ion-transport, gramicidin A channel activity in planar lipid bilayers was investigated in the presence of calcium and annexins II, III or V. The experiments were performed with membranes consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in 300 mM KCl solution buffered to pH 7.4 and with either 0.1 or 1 mM calcium added to the solution. Annexin (1 microM) was subsequently applied to the cis side of the membrane. All three annexins (II, III and V) when tested at 1 mM calcium decreased the gramicidin single-channel conductance. Annexins II and III increased the mean lifetime of the channels whereas annexin V seemed to have no influence on the mean lifetime. Since the lifetime of gramicidin A channels is a function of the rate constant for dissociation of the gramicidin dimer, which is dependent on the physical properties of the lipid phase, binding of annexins II and III seems to stabilize the gramicidin channel owing to a change of the bilayer structure.

The effect of annexins II, III and V, purified from different species, on the calcium-activated chloride current across the stage-V to stage-VI Xenopus laevis oocyte membrane was tested either directly, using calcium entry mediated by depolarization, by A23187 permeabilization of oocytes or indirectly by quisqualate stimulation of a metabotropic glutamate receptor in the membrane expressed by the oocyte after injection of mRNA. The annexins isolated from the Ehrlich ascites cell, which is a mouse tumor cell, were found to be potent inhibitors of the chloride current, showing half-maximal inhibition at 50 nM, whereas no block was found using bovine or porcine annexins isolated from lung tissue. Of the annexins tested, we found annexin III to be naturally occurring in the oocyte, while only trace amounts of annexins II and V could be demonstrated. The inhibition pattern varied somewhat according to the stimulus method, the inhibition being more complete when an indirect stimulus via the metabotropic receptor was applied compared to a direct calcium stimulus.