ACTIN-DEPENDENT ACTIVATION OF ION CONDUCTANCES IN BRONCHIAL EPITHELIAL-CELLS

Activation of Cl- and K+ channels is necessary to drive ion secretion in epithelia. There is substantial evidence from previous reports that vesicular transport and exocytosis are involved in the regulation of ion channels. In the present study we examined the role of cytoskeletal elements and components of intracellular vesicle transport on ion channel activation in bronchial epithelial cells. To this end, cells were incubated with a number of different compounds which interact with either microtubules or actin microfilaments, or which interfere with vesicle transport in the Golgi apparatus. The effectiveness of these agents was verified by fluorescence staining of cellular microtubules and actin. The function was examined in Cl-36(-) efflux studies as well as in whole-cell (WC) patch-clamp and cell-attached studies. The cells were studied under control conditions and after exposure to (in mmol/l) ATP (0.1), forskolin (0.01), histamine (0.01) and hypotonic bath solution (HBS, NaCl 72.5). In untreated control cells, ATP primarily activated a K+ conductance whilst histamine and forskolin induced mainly a Cl- conductance. HBS activated both K+ and Cl- conductances. Incubation of the cells with brefeldin A (up to 100 mu mol/l) did not inhibit WC current activation and Cl-36(-) efflux. Nocodazole (up to 170 mu mol/l) reduced the ATP-induced WC current, and mevastatin (up to 100 mu mol/l) the cell-swelling-induced WC current. Neither had any effect on the WC current induced by forskolin and histamine. Also Cl-36(-) efflux induced by HBS, ATP, forskolin and histamine was unaltered by these compounds. Similarly, colchicine (10 mu mol/l) and taxol (6 mu mol/l) affected neither Cl-36(-) efflux nor WC current induced by ATP, forskolin, histamine or HBS. In contrast, depolymerisation of actin by cytochalasin D (10 mu mol/l) significantly attenuated Cl-36(-) effluxes and WC current activation by the above-mentioned agonists. Incubation with a C2 clostridial toxin (5 nmol/l) showed similar effects on WC currents. Moreover, when cytochalasin D (10 mu mol/l), C2 clostridial toxins (5 nmol/l), or phalloidin (10 mu mol/l) were added to the pipette filling solution current activation was markedly reduced. However, in excised inside-out membrane patches, cytochalasin D (10 mu mol/l), G-actin (10 mu mol/l) and phalloidin (10 mu mol/l) had no effect. These data suggest that actin participates in the activation of ion channels in 16HBE14o- epithelial cells and support the concept that exocytosis is a crucial step in the regulation of Cl- and K+ channels in these cells.