CHARACTERIZATION OF 2 DISTINCT CL- CONDUCTANCES IN FUSED HUMAN RESPIRATORY EPITHELIAL-CELLS .1. ANION SELECTIVITIES, STIMULATION AND INTERMESHING SIGNAL-TRANSDUCTION PATHWAYS

With the aim of further elucidating the role of the epithelial Cl- con ductance and its defect in cystic fibrosis (CF) patients we studied th e properties and regulation of the Cl- conductance in primary cultures of human nasal polyp epithelia. To facilitate microelectrode puncture s and to gain access to the cytoplasmic compartment for injection of a ntibodies, we prepared giant cells using a polyethylene-glycol fusion technique. The membrane potential (V-m) and resistance (R(m)) and thei r responses to ionic substitutions in the bath were measured under con trol conditions and in the presence of different secretagogues. In non -CF cells V-m averaged -12.5 mV (SD +/- 6.6 mV, n = 69) and was indepe ndent of time after fusion, while R(m) dropped from 12.4 +/- 7.3 M Ome ga (n = 51) to 3.5 +/- 5.5 M Omega (n = 24) in the 2nd week after fusi on. The low V-m values reflected a vanishing K+ conductance in the pre sence of a dominating Cl- conductance that increased with time. In you ng cells, a Cl- conductance prevailed which could be stimulated by app lication of the Ca2+ ionophore, A23187, or of carbachol. As determined in CF cells, it had an outwardly rectifying current/voltage (i/V) rel ationship and exhibited the selectivity sequence I- > Br- > Cl- > F- > isethionate (ISE(-)) both in V-m and R(m) measurements. With increasi ng age after fusion, a Cl- conductance prevailed in non-CF cells which could be stimulated by cyclic adenosine monophosphate (cAMP) or forsk olin and which was downregulated by A23187. It had a linear i/V relati onship and exhibited the selectivity sequence Br- > Cl- > I- > F- > IS E(-) if determined from V, measurements, but a sequence of Cl- > Br- > F- = ISE(-) > I- if determined from R(m) measurements. This points to multiple-ion pore behaviour of the respective Cl- channel. In agreeme nt with observations described in the following publication, the resul ts suggest that the cAMP-regulated Cl- conductance corresponds to the CF-gene product while the molecular nature of the Ca2+-regulated Cl co nductance is not yet known.