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Human airway epithelia are predominantly Na(+)-absorbing epithelia. To investigate the mechanisms for Na+ absorption across airway epithelia, the driving forces and paths for Na+ translocation across each membrane were examined with double-barreled Na(+)-selective microelectrodes in cultured human nasal epithelium (HNE). Under control conditions, intracellular Na+ activity (acNa) was 23 +/- 1 mM (n = 44 preparations, 393 impalements). Amiloride (10(-4) M) hyperpolarized the apical membrane and increased the fractional apical membrane resistance but did not affect acNa. Exposure to Na(+)-free luminal solution induced bioelectric responses similar to amiloride but also reduced acNa to 8 +/- 1 mM. Reduction of luminal Na+ concentration ([Na+]) in the presence of amiloride also reduced acNa without further changes in bioelectric parameters. Reduction of serosal [Na+] decreased aNac, a response blocked by bumetanide (10(-4) M). Ouabain (10(-4) M, serosal) led to a reduction in equivalent short-circuit current (Ieq) and increase in acNa. We conclude that 1) acNa is higher in HNE than in most mammalian epithelial cells, 2) the apical membrane expresses a conductive Na+ path, and 3) the basolateral membrane transports Na+ via the Na(+)-K(+)-adenosinetriphosphatase and a Na(+)-K(+)-2Cl- cotransport system.

Cystic fibrosis (CF) airway epithelia exhibit raised transepithelial Na+ transport rates, as determined by open-circuit isotope fluxes and estimates of the amiloride-sensitive equivalent short-circuit current (Ieq). To study the contribution of apical and basolateral membrane paths to raised Na+ transport in CF, CF nasal epithelial cultures were studied with double-barreled Na(+)-selective microelectrodes and the Ussing chamber technique. Intracellular Na+ activity (acNa) was 24.1 +/- 1.5 mM (n = 36), a value similar to acNa of normal nasal epithelial cells. Reduction of luminal [Na+] to 3 mM abolished Ieq and reduced acNa. Amiloride (10(-4) M) abolished Ieq but increased acNa from 20 +/- 2 to 36 +/- 7 mM (n = 10). Amiloride-induced increase in acNa was not affected by serosal [Na+] reduction but was blocked by preexposure to reduced luminal [Na+]. Amphotericin B increased Ieq during amiloride exposure, indicating that amiloride did not inhibit NA(+)-K(+)-ATPase. Ouabain abolished Ieq and slowly raised acNa. Reduction of serosal [Na+] led to a decrease in acNa that was blocked by bumetanide. It is concluded that 1) CF airway epithelia exhibit an increased apical membrane Na+ permeability, 2) acNa is regulated to a normal level in CF cells despite increased transcellular Na+ fluxes, 3) the abnormal increase in acNa in response to amiloride is dependent on luminal Na+, 4) Na+ is transported across the basolateral membrane by a bumetanide-sensitive cotransport mechanism, and 5) ouabain inhibits the basolateral Na(+)-K(+)-ATPase, causing slow dissipation of the chemical and electrical gradients across the cell membranes.

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.

A method for determination of shunt resistance (Rs) and absolute conductive ion permeabilities of the apical membrane in epithelia from steady-state data is described. The method assumes that the currents are satisfactorily described by the Goldman-Hodgkin-Katz regime. Its application requires measurements of standard transepithelial electrophysiological parameters and of one or more intracellular ion activities. It is applicable under both open- and short-circuit conditions. The method was tested in an electrophysiological analysis of cultured normal and cystic fibrosis (CF) human nasal epithelium. In 15 normal and 10 CF preparations with mean transepithelial resistances of 338 and 427 omega.cm2, Rs was 412 and 623 omega.cm2, respectively. The Rs values determined with the present method were strongly correlated (r = 0.94) with those obtained with another method available in the electrophysiological literature but were as a mean 20% lower. Amiloride increased Rs by 25% in CF and by 8% in normal preparations. In normal preparations, the apical Cl permeability (PCla) was 3.6 x 10(-6) cm/s, and the apical Na permeability (PNaa) was 1.6 x 10(-6) cm/s. In CF preparations, PCla was reduced to a maximum of 2.3 x 10(-7) cm/s, whereas PNaa was increased to 6.2 x 10(-6) cm/s. The apical membrane electromotive force was -1 mV in normal and 43 mV in CF preparations. It is concluded that the method can be used to calculate Rs, apical membrane ion permeabilities, and electromotive forces from steady-state electrophysiological data.

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.

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.