More acidic ASL on cultured primary human CF vs. normal bronchial epithelium. One hundred microliters of KBR was applied to the apical surface of cultures of CF and NL bronchial epithelium. Aliquots of liquid (1-5 μl, quadruplicate samples from cultures of 20 CF patients and 24 NL patients) were sampled at intervals and assayed for pH (A) and (B). *, P < 0.001 pH_ASL CF vs. NL.

H⁺,K⁺-ATPase in cultured and freshly excised human bronchial epithelium. (A) Immunostaining of well differentiated primary bronchial cultures (Upper) and freshly excised bronchus (Lower) were analyzed with confocal microscopy. Specimens were stained with a monoclonal antibody recognizing the β-subunit of the ATPase, followed by a Texas Red-labeled secondary antibody and fluorescein-labeled phalloidin. Red and green channels were registered by sequential scanning in the xz axis (cultures; magnification, ×200) and xy axis (bronchial sections; magnification, ×80). H⁺,K⁺-ATPase is heavily expressed in the apical membrane of ciliated cells of the bronchial cultures, as well as in superficial epithelium and ciliated ducts in the human bronchus. Actin cytoskeleton (phalloidin) indicates the position of the plasma membrane. L, lumen; S, support; D, ciliated duct. (B) Ouabain, not Sch28080, inhibited K⁺-dependent ASL acidification. Fifty microliters of KBR containing 0 mM or 20 mM K⁺ was applied apically to NL bronchial epithelial cultures in the presence or absence of inhibitors. Ouabain (500 μM), an inhibitor of the nongastric isoform of the H⁺,K⁺-ATPase, and Sch28008 (10 μM), an inhibitor of the gastric isoform of the H⁺,K⁺-ATPase, were added. pH_ASL and K⁺_ASL were monitored over 90 min. *, P < 0.001 pH_ASL control vs. time_zero; †,pH_ASL sch28080 vs. Time_zero.(C) mRNA expression of the nongastric isoform of the H⁺,K⁺-ATPase was detected by RT-PCR in cultured bronchial epithelia from three NL subjects as well as in normal colonic, but not gastric, tissue.

The paracellular path conducts and exhibits selectivity for individual anions. Primary CF epithelial cultures were mounted in Ussing chambers. Inhibition of ENaC (Amiloride, 10^-4 M) eliminated significant apical membrane conductance because these cultures also lack CFTR. Changes in potential difference, under these circumstances, thus reflect paracellular ion movement. The basolateral side was perfused with KBR. The changes in transepithelial potential difference (ΔPD) result from switching the apical solution from KBR to one where the chloride and bicarbonate concentrations were inverted (imposing equal “basolateral-to-apical” chloride and “apical-to-basolateral” bicarbonate gradients), and subsequently to one where 95 mM gluconate replaced 95 mM bicarbonate in the apical solution (i.e., imposing equal basolateral-to-apical chloride and apical-to-basolateral gluconate gradients). Results are shown as the PD. Results from duplicate cultures of nine CF epithelia were analyzed. *, P < 0.05 PD apical “high apical bicarbonate” solution vs. KBR. **, P < 0.05 apical “high gluconate” vs. “high bicarbonate” and KBR.

CF and NL cultures exhibit K⁺-dependent acidification and ASL K⁺ depletion. One hundred microliters of KBR (0, 5, or 20 mM/liter [K⁺]) was applied to the apical surface of CF and NL bronchial epithelial cultures. Microaliquots of apical liquid were assayed for pH. NL (A) and CF (B) cultures exhibit -dependent acidification over 6 h. †, P < 0.0001, 0 mM vs. 20 mM K⁺; *, P < 0.01, 0 mM vs. 5 mM K⁺. (C) Acidification rates measured in CF and 24 NL cultures in response to solutions containing 0, 5, and 20 mM K⁺ measured over 6 h. *, P < 0.01 rates of ASL acidification on CF vs. NL cultures at each [K⁺]. (D)[K⁺]_ASL depletion by CF and NL cultures. One hundred microliters of KBR ([K⁺] 20 mM/liter) was applied to the apical surface of cultures of CF and NL bronchial epithelia. Microaliquots of apical liquid were assayed for [K⁺]. P = 0.56.

Increased intracellular cAMP alkalinizes ASL on NL bronchial cultures and acidifies ASL on CF bronchial cultures. We tested the effect of cAMP activation (10^-5 M forskolin/200 μM IBMX bilaterally) on pH_ASL regulation in NL (Fig. 5A), disease control (Fig. 5B), and CF (Fig. 5C) bronchial epithelial cultures. One hundred microliters of bicarbonate/K⁺-free saline Ringer's solution (pH 5.6) was applied apically. Microaliquots of apical liquid were sampled and assayed for pH_ASL.(A) *, P < 0.001 NL control vs. NL forskolin/IBMX. (B) *, P < 0.001 disease control vs. disease control + forskolin/IBMX. (C) *, P < 0.005 CF control vs. CF forskolin/IBMX.

Transepithelial movement after ASL acid-challenge is reduced in CF vs. NL cultures. One hundred microliters of saline Ringer's solution (pH 3.3) was applied apically to NL and CF bronchial epithelial cultures. The basolateral medium was KBR or similar solution where 25 mM Na⁺ Hepes replaced 25 mM Na⁺ , pH both = 7.45. Recovery from luminal acid challenge in the presence and absence of basolateral is shown for NL (A) and CF (B). A significantly greater basolateral dependence is observed in NL vs. CF cultures.