Tsutomu Tamada, Martin J Hug, Raymond A Frizzell, Robert J Bridges
Calu-3 cells secrete HCO3 - in response to cAMP agonists but can be stimulated to secrete Cl- with K + channel activating agonists. Microelectrode and impedance analysis experiments were performed to obtain a better understanding of the conductances and driving forces involved in these different modes of anion secretion in Calu-3 cells. Microelectrode studies revealed apical and basolateral membrane depolarizations upon the addition of forskolin (Vap –52 mV vs. –21 mV; Vbl –60 mV vs. –44 mV) that paralleled the hyperpolarization of the mucosal negative transepithelial voltage (VT –8 mV vs. –23 mV). These changes were accompanied by a decrease in the apical membrane fractional resistance (FRap) from approximately 0.50 to 0.08, consistent with the activation of an apical membrane conductance. The subsequent addition of 1-ethyl-2-benzimidazolinone (1- EBIO), a K+ channel activator, hyperpolarized Vap to – 27 mV, Vbl to – 60 mV and VT to – 33 mV. Impedance analysis revealed the apical membrane resistance (Rap) of the forskolinstimulated cells was less than 20 W cm2 , indeed in most monolayers Rap fell to less than 5 W cm2 . The impedance derived estimate of the basolateral membrane resistance (Rbl) was approximately 170 W cm2 in forskolin treated cells and fell to 50 W cm2 with the addition of 1-EBIO. Using these values for the Rbl and the FRap value of 0.08 yields a Rap of approximately 14 W cm2 in the presence of forskolin and 4 W cm2 in the presence of forskolin plus 1-EBIO. Thus, by two independent methods, forskolinstimulated Calu-3 cells are seen to have a very high apical membrane conductance of 50 to 200 mS/cm2 . Therefore, we would assert that even at one-tenth the anion selectivity for Cl- , this high conductance could support the conductive exit of HCO3 - across the apical membrane. We further propose that this high apical membrane conductance serves to clamp the apical membrane potential near the equilibrium potential for Cl- and thereby provides the driving force for HCO3 - secretion in forskolinstimulated Calu-3 cells. The hyperpolarization of Vap and Vbl caused by 1-EBIO provides a driving force for Cl- exit across the apical membrane, inhibits the influx of HCO3 - on the Na+ :HCO3 - cotransporter across the basolateral membrane, activates the basolateral membrane Na+ :K:2Cl- cotransporter and thereby provides the switch from HCO3 - secretion to Clsecretion.
