Cl- transport was studied in human nasal epithelium, a predominantly Na+-absorbing proximal airway epithelium. Intracellular Cl- activity (aClc) and the electrical potentials across the apical (Va) and basolateral (Vb) membranes were measured with double-barreled, Cl- -selective microelectrodes to characterize the driving forces for Cl- flow across each membrane. Under control conditions (bilateral Krebs-bicarbonate Ringer), Va was -26.1 +/- 1.2 mV, Vb was -36.2 +/- 1.2 mV, and aCL(c) was 42.7 +/- 2.0 mM (n = 34), indicating that Cl- is near electrochemical equilibrium across the apical membrane but significantly above equilibrium across the basolateral membrane. Reduction of luminal [Cl-] from 120 to 3 mM reduced aClc from 42.7 +/- 4.0 to 27.0 +/- 3.5 mM, depolarized Va, and increased fractional apical membrane resistance (fRa) and transepithelial resistance (Rt). Serosal bumetanide reduced aClc by 10 mM without affecting electrical parameters. Reduction of serosal [Cl-] from 120 to 3 mM resulted in a rapid decrease in Vb, a decrease in fRa and an increase in Rt. Also, serosal [Cl-] reduction led to a slow decrease in aClc rom 45.5 +/- 2.5 to 31.1 +/- 4.2 mM) that could be inhibited by bumetanide. The data are consistent with the following conclusions: 1) Cl- is transported across the apical membrane through a conductive pathway; and 2) Cl- is translocated across the basolateral membrane by an electrically silent bumetanide-sensitive cotransport system and by a minor conductive path.

The response of cultured human nasal epithelia to hypertonic bathing solutions was tested using ion-selective microelectrode and quantitative microscopy. Raised luminal, but not serosal, osmolality (±150 mM mannitol) decreased Na' absorption but did not induce Cl-secretion. Raised luminal osmolality increased cell Cl-activity, Na' activity, and transepithelial resistance and decreased both apical and basolateral membrane potentials and the fractional resistance of the apical membrane; equivalent circuit analysis revealed increases in apical, basolateral, and shunt resistances. Prolonged exposure (10 min) to 430 mosM luminal solution elicited no regulation of any parameter. Optical measurements revealed a reduction in the thickness of preparations only in response to luminal hypertonic solutions. We conclude that (a) airway epithelial cells exhibit asymmetric water transport properties, with the apical membrane water permeability exceeding that of the basolateral membrane; (b) the cellular response to volume loss is a deactivation of the basolateral membrane K+ conductance and the apical membrane Cl-conductance; (c) luminal hypertonicity slows the rate of Na' absorption but does not induce Cl-secretion; and (d) cell volume loss increases the resistance of the paracellular path. We speculate that these properties configure human nasal epithelium to behave as an osmotic sensor, transducing information about luminal solutions to the airway wall. (J. Clin. Invest. 1994. 94:779-787.)

Cystic fibrosis (CF) airway epithelia express a defect in adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of apical membrane Cl- channels. Recent patch-clamp studies have raised the possibility that Ca2+ -dependent mechanisms for the activation of Cl- secretion may be preserved in CF airway epithelia. To determine 1) whether intact normal (N1) and CF airway epithelia exhibit a Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanisms initiate Cl- secretion via activation of an apical membrane Cl- conductance (GCl-), nasal epithelia from N1 and CF subjects were cultured on collagen membranes, and responses to isoproterenol or Ca2- ionophores [A23187 10(-6) M; ionomycin (10(-5)M)] were measured with transepithelial and intracellular techniques. Isoproterenol induced activation of an apical membrane GCl- in N1 cultures but was ineffective in CF. In contrast, in both N1 and CF amiloride-pretreated cultures, A23187 induced an increase in the equivalent short-circuit current that was associated with an activation of an apical membrane Gc1- and was bumetanide inhibitable. A23187 addition during superfusion of the lumen with a low Cl- (3 mM) solution reduced intracellular Cl- activity of CF cells. A Ca2+ ionophore of different selectivity properties, ionomycin, was also an effective Cl- secretagogue in both N1 and CF cultures. We conclude that 1) the A23187 induced Cl- secretion via activation of an apical GCl- in N1 human nasal epithelium, and 2) in contrast to an isoproterenol-dependent path, a Ca2+ -dependent path for GCl- activation is preserved in CF epithelia.

Planar silicon chips with 1-2-microm etched holes (average resistance: 2.04 +/- 0.02 MOmega in physiological buffer, n = 274) have been developed for patch-clamp recordings of whole-cell currents from cells in suspension. An automated 16-channel parallel screening system, QPatch 16, has been developed using this technology. A single-channel prototype of the QPatch system was used for validation of the patch-clamp chip technology. We present here data on the quality of patch-clamp recordings and from actual drug screening studies of human potassium channels expressed in cultured cell lines. Using Chinese hamster ovary (CHO) and human embryonic kidney cells (HEK), gigaseals of 4.1 +/- 0.4 GOmega (n = 146) and high-quality whole-cell current recordings were obtained from hERG and KCNQ4 potassium channels. Success rates for gigaseal recordings varied from 40 to 95%, and 67% of the whole-cell configurations lasted for >20 min. Cells were maintained in suspension up to 4 h in a cell storage facility that is integrated in the QPatch 16. No decline in patchability was observed during this time course. A series of screens was conducted with known inhibitors of the hERG and KCNQ4 potassium channels. Dose-response relationship characterizations of verapamil and rBeKm-1 blockage of hERG currents provided IC(50) values similar to values reported in the literature.

Cultured human nasal epithelia derived from cystic fibrosis (CF) patients were studied with double-barreled, Cl- -selective microelectrodes to measure membrane potentials and intracellular Cl- activity (aClc). The aClc of CF cultures was 46.5 +/- 2.5 mM (n = 28), a value not significantly different from aClc of normal human nasal cells. Reduction of the luminal [Cl-] from 120 to 3 mM failed to reveal any apical Cl- permeability (conductive or nonconductive) in CF cultures. Bumetanide (10(-4) M, serosal) led to a 10 mM decrease in aClc without affecting the electrical parameters of the cells. Reduction of serosal [Cl-] led to a marked decrease in aClc (from 58.0 +/- 6.7 to 26.8 +/- 2.9 mM) that could partly be blocked by bumetanide. Reduction of serosal [Cl-] led to a rapid depolarization (5.4 +/- 0.7 mV) of the basolateral membrane potential (Vb), a decrease of the fractional apical membrane resistance (0.03 +/- 0.01), and an increase (34 +/- omega.cm2) in the transepithelial resistance (Rt). We conclude that 1) the apical membrane of CF airway epithelia is impermeable to Cl-, and 2) Cl- transport across the basolateral membrane occurs mainly through a bumetanide-inhibitable cotransport system but also through a Cl- conductance, neither of which appears to be affected by CF.