The electrophysiologic properties of ALI cultures of NPSE and CSE cells were measured as previously described.10 Briefly, Snapwell cultures were mounted in Ussing chambers (Physiologic Instruments, La Jolla, Calif). Each bath contained warmed (37°C) Krebs bicarbonate Ringer solution (containing 140mM Na+, 120mM Cl−, 5.2mM K+, 1.2mM Ca2+, 1.2mM Mg2+, 2.4mM HPO42+, 0.4mM H2PO4−, 25mM HCO3−, and 5mM glucose), circulated by gas lift with 95% oxygen and 5% carbon dioxide. Bioelectric properties were digitally recorded from the output of voltage clamps (Physiologic Instruments) using a software program (ACQUIRE; Physiologic Instruments). The transepithelial voltage was clamped to 0 mV, and the short circuit current (Isc) was continuously recorded. To measure basal bioelectric properties and rates of Na+ absorption and Cl− secretion, tissues were mounted and Isc and potential difference were recorded after Isc stabilized (mean ± SD, 14.8 ± 1.0 minutes). Transepithelial resistance (Rt) was calculated using Ohm's law. Low-resistance tissues (R<300 Ω·cm2) were excluded from the study. In one protocol, an epithelial Na+ channel inhibitor, amiloride hydrochloride (100μM) (Sigma-Aldrich Corp), was added to the apical bath to measure basal Na+ transport rates. In a second protocol, an inhibitor of the Na+-K−-2Cl− channel, bumetanide (100μM) (Sigma-Aldrich Corp), was added to the basolateral bath to measure the Na+-K+-2Cl− cotransport system–mediated current, that is, basal Cl− transport. In this protocol, after 20 minutes, amiloride was added to measure Na+ transport, and a purinergic receptor agonist, uridine triphosphate (UTP), 100μM (Sigma-Aldrich Corp), which ultimately raises intracellular Ca2+ levels, was added to the apical bath to assess Ca2+-activated Cl− secretion. Two additional protocols were designed to also explore regulation of Cl− transport. First, the cyclic adenosine monophosphate–stimulating agent forskolin (10μM) (Sigma-Aldrich Corp) was added bilaterally to activate cystic fibrosis transmembrane conductance regulator, followed by 100μM UTP and, finally, 100μM bumetanide. Second, after pretreatment with 100μM amiloride, 100μM UTP was added to the apical bath, and then 10μM forskolin was added bilaterally. Separate culture preparations for each patient were included in each protocol.