Prior studies have shown that fluid secretions from airway submucosal glands in cystic fibrosis (CF) are reduced and hyperviscous, possibly contributing to the pathogenesis of CF airway disease. Because the CF transmembrane conductance regulator (CFTR) protein can transport both chloride and bicarbonate, we investigated whether gland fluid pH is abnormal in early CF, using nasal biopsies from pediatric subjects having minimal CF lung disease. Gland fluid pH, measured in freshly secreted droplets under oil stained with BCECF-dextran, was 6.57 +/- 0.09 (mean +/- SE) in biopsies from six CF subjects, significantly lower than 7.18 +/- 0.06 in eight non-CF biopsies (P < 0.01). To rule out the possibility that the apparent gland fluid hyperacidity in CF results from modification of fluid pH by the airway surface, a microcannulation method was used to measure pH in fluid exiting gland orifices. In pig trachea and human bronchi, gland fluid pH was reduced by up to 0.45 units by CFTR inhibitors, but was not affected by amiloride. Acid base transport in the surface epithelium of pig trachea was studied from pH changes in 300-nl fluid droplets deposited onto the oil-covered airway surface. The droplets had specified ionic composition/pH and/or contained transporter activators/inhibitors. We found evidence for CFTR-dependent bicarbonate transport by the tracheal surface epithelium as well as ATP/histamine-stimulated proton secretion, but not for sodium/proton or chloride/bicarbonate exchange. These results provide evidence for intrinsic hyperacidity in CF gland fluid secretions, which may contribute to CF airway pathology.

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The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated epithelial Cl− channel that, when defective, causes cystic fibrosis. Screening of a collection of 100,000 diverse small molecules revealed four novel chemical classes of CFTR inhibitors with Ki < 10 μM, one of which (glycine hydrazides) had many active structural analogues. Analysis of a series of synthesized glycine hydrazide analogues revealed maximal inhibitory potency for N-(2-naphthalenyl) and 3,5-dibromo-2,4-dihydroxyphenyl substituents. The compound N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101) reversibly inhibited CFTR Cl− conductance in <1 min. Whole-cell current measurements revealed voltage-dependent CFTR block by GlyH-101 with strong inward rectification, producing an increase in apparent inhibitory constant Ki from 1.4 μM at +60 mV to 5.6 μM at −60 mV. Apparent potency was reduced by lowering extracellular Cl− concentration. Patch-clamp experiments indicated fast channel closures within bursts of channel openings, reducing mean channel open time from 264 to 13 ms (−60 mV holding potential, 5 μM GlyH-101). GlyH-101 inhibitory potency was independent of pH from 6.5–8.0, where it exists predominantly as a monovalent anion with solubility ∼1 mM in water. Topical GlyH-101 (10 μM) in mice rapidly and reversibly inhibited forskolin-induced hyperpolarization in nasal potential differences. In a closed-loop model of cholera, intraluminal GlyH-101 (2.5 μg) reduced by ∼80% cholera toxin–induced intestinal fluid secretion. Compared with the thiazolidinone CFTR inhibitor CFTRinh-172, GlyH-101 has substantially greater water solubility and rapidity of action, and a novel inhibition mechanism involving occlusion near the external pore entrance. Glycine hydrazides may be useful as probes of CFTR pore structure, in creating animal models of CF, and as antidiarrheals in enterotoxic-mediated secretory diarrheas.