ISSN:
1432-2013
Keywords:
Human respiratory epithelial cells
;
CF tissue
;
Intracellular microinjection
;
CFTR antibody
;
Cytosolic Cl− channel inhibitor
;
Ca2+-regulated
;
Cl− conductance
;
cAMP-regulated
;
Cl− conductance
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Abstract The present microelectrode experiments on fused respiratory epithelial cells of cystic fibrosis (CF) origin and non-CF origin aim at characterizing the molecular basis of the Cl− conductances regulated by cyclic adenosine monophosphate (cAMP) or respectively Ca2+, as described in the preceding publication. Cell membrane potential (V m) and resistance (R m) were recorded as well as their response to substitution of 90% of bath Cl− by isethionate (ΔV m,ISE), by I− (ΔV m,I), or by other halide anions. Fused CF cells had significantly (P〈0.05) higher control V m values (P−18.0 ±9.4 mV, ±SD, n=68) than fused non-CF cells (−12.5±6.6 mV, n=69) and responded to the Ca2+ ionophore A23187 with an increase in the V m response to Cl− substitution, but did not respond to forskolin. This indicates that CF cells express only the Ca2+-stimulated Cl− conductance. Injection of the antibody M3A7 against a fusion protein containing amino acids 1195 to 1480 of the CF gene product into young, forskolin-stimulated or old non-CF cells decreased ΔV m,ISE and ΔV m,I within 15 min to values observed in CF cells. This indicates inhibition of the cAMP-stimulated Cl− conductance and supports the molecular identity of this conductance with the CF gene product. However, the slow onset of inhibition does not allow secondary effects to be excluded and a slight fall in R m remains unexplained. Stimulation of the Ca2+-regulated Cl− conductance was not impaired. Injection of M3A7 into CF cells or of a control antibody in non-CF cells had no effect. In the search for the single-channel equivalent of the Ca2+-stimulated Cl− conductance we injected a concentrated placental cytosol fraction containing a cytosolic inhibitor of the outwardly rectifying intermediate conductance (ORIC) Cl− channel into fused non-CF cells stimulated either with A23187 or forskolin. However, no effect was observed. This speaks against a role of the ORIC Cl− channel in the Ca2+-activated Cl− conductance, although it cannot definitely be excluded.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00374657
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