Human Bronchial Cell Line 16HBE14o Research Articles

Modulation of calcium-dependent chloride secretion by basolateral SK4-like channels in a human bronchial cell line.

The human bronchial cell line16HBE14o- was used as a model of airway epithelial cells to study the Ca(2+)-dependent Cl(-) secretion and the identity of K(Ca) channels involved in the generation of a favorable driving force for Cl(-) exit. After ionomycin application, a calcium-activated short-circuit current ( I(sc)) developed, presenting a transient peak followed by a plateau phase. Both phases were inhibited to different degrees by NFA, glybenclamide and NPPB but DIDS was only effective on the peak phase. (86)Rb effluxes through both apical and basolateral membranes were stimulated by calcium, blocked by charybdotoxin, clotrimazole and TPA. 1-EBIO, a SK-channel opener, stimulated (86)Rb effluxes. Block of basolateral K(Ca) channels resulted in I(sc) inhibition but, while reduced, I(sc) was still observed if mucosal Cl(-) was lowered. Among SK family members, only SK4 and SK1 mRNAs were detected by RT-PCR. KCNQ1 mRNAs were also identified, but involvement of K(cAMP) channels in Cl(-) secretion was unlikely, since cAMP application had no effect on (86)Rb effluxes. Moreover, chromanol 293B or clofilium, specific inhibitors of KCNQ1 channels, had no effect on cAMP-dependent I(sc). In conclusion, two distinct components of Cl(-) secretion were identified by a pharmacological approach after a Cai2+ rise. K(Ca) channels presenting the pharmacology of SK4 channels are present on both apical and basolateral membranes, but it is the basolateral SK4-like channels that play a major role in calcium-dependent chloride secretion in 16HBE14o- cells.

Effects of hydrogen peroxide and hydroxyl radicals on the cytosolic side of a non-selective cation channel in the cultured human bronchial epithelial cell line 16HBE14o-.

Respiratory tissues can be damaged by the exposure of airway epithelial cells to reactive oxygen species (ROS) that generate an oxidative stress. We studied the effects of the hydroxyl radical *OH, for which there is no natural intra- or extracellular scavenger, on a Ca2+-activated, non-selective cation channel (NSC(Ca)) which might participate in the transepithelial Na+ fluxes involved in the maintenance of the cytosolic [Na+] ([Na+]i). We identified and characterized NSC(Ca) in inside-out excised membrane patches from cells of the human bronchial cell line 16HBE14o- and exposed the cytoplasmic side of NSC(Ca) to H2O2 or *OH created in front of the patch pipette. In these cells, the NSC channel was Ca2+ (above 0.1 microM) and voltage dependent. The channel's low open probability ( P(o)) at hyperpolarized voltages increased rapidly to a very high value upon short exposure (seconds) to *OH or to H2O2 in a bath solution containing millimolar [Ca2+]. This sensibility to ROS was reversible on wash-out of the oxidants. Long exposure (several minutes) to *OH in a bath solution containing 10(-7) M Ca2+ activated the channel irreversibly, a finding that has pathophysiological implications. The functioning of the NSC(Ca) protein channel may be modified by the intracellular redox status and the reversibility of the sensitivity to ROS depends on [Ca2+]i. The role of NSC(Ca) channels in airway epithelia is not yet understood, but might conform to what might be expected of an NSC channel: increase in [Na]i following intracellular exposure to *OH or H2O2. Reversible or irreversible excessive opening of cation channels may induce gradual cellular responses, including changes in Na+/K+-ATPase activity, cell volume regulation, membrane polarization, activation of transcription factors required for the expression of genes of cytokines or chemokines, apoptosis and necrosis, thus triggering inflammation.

Effects of hydroxyl radicals on outwardly rectifying chloride channels in a cultured human bronchial cell line (16HBE14o-).

Respiratory pathologies can result from the exposure of airway epithelial cells to oxidative stress. We studied the effects of the hydroxyl radical *OH, for which there is no natural intra- or extracellular scavenger, on an outwardly rectifying chloride channel (ORCC). In the human bronchial cell line 16HBE14o-, the cytoplasmic side of ORCC in inside-out excised membrane patches was exposed to *OH created by simultaneously superfusing Fe2+ and H2O2 in front of the patch-pipette. ORCC was activated by depolarizing voltage steps. Its open probability (Po) increased with bath [Ca2+] above 1 microM. Upon brief exposure to *OH, ORCC first closed and then alternated between periods of closure and normal activity. The duration of closure increased with the duration of *OH exposure but voltage steps could reopen the channel. After 10 min exposure to *OH, however, the channel closed irreversibly, regardless of the number of subsequent voltage steps or the duration of washing. Low [Ca2+] in the bath accelerated the irreversible closure of the channel in the presence of *OH. Intracellular application of *OH progressively inhibited ORCC activity by inducing long closure periods that increased with time. This might have important pathophysiological implications in the process of inflammation.