Bioelectric effects of quinine on polarized airway epithelial cells

Abstract

Quinine has been increasingly utilized as a placebo in cystic fibrosis (CF) clinical trials, including those leading to FDA approval of inhaled tobramycin, recent studies of anti-inflammatory aerosols such as glutathione, and clinical testing of hypertonic saline aerosols to augment mucous clearance. The drug effectively masks taste of experimental therapeutics, but could also confer changes in processes contributing to CF pathogenesis, including chloride secretion and paracellular ion permeability. In the Ussing chamber, concentrations of quinine (1 mg/ml) anticipated in the airways of CF subjects after aerosolization led to changes in chloride transport in Calu-3 (airway serous glandular) cell monolayers. Tissue resistance was significantly disrupted by the compound in both Calu-3 and primary airway epithelial cells in vitro. Lower doses of quinine (between 10 and 100 microg/ml) strongly inhibited the chloride secretory mechanism that utilizes CFTR, and forskolin activated I(SC) was reduced by approximately 24% and 44% in the presence of 10 and 100 microg/ml quinine, respectively. Our findings indicate that quinine disrupts airway epithelial functional integrity and blocks transepithelial chloride transport. The use of quinine as a taste-masking agent may have bioelectric effects relevant to CF trials using aerosolized drug delivery.

Figure 1. Apical addition of high dose quinine to Calu-3 monolayers

Cells were grown at an air-liquid interface and studied in Ussing chambers. A: Calu-3 cells in Ringers solution followed by 1) mucosal (M) low- Cl⁻ solution, 2) addition of 1 mg/mL quinine, and 3) addition of 200 μM glybenclamide. B: Same experiment as in A, with mucosal and serosal low- Cl⁻ solution (i.e. no chloride gradient) prior to addition of quinine.

Figure 2. Quinine increases Cl⁻ permeability in airway epithelia through a paracellular pathway associated with decreased epithelial resistance

A: In Calu-3 monolayers, apical quinine (1 mg/mL) increased the monolayer permeability (19.4 ± 3.4 μA/cm², as also shown in Figure 1A,* p < 0.001 compared with untreated controls, n = 16, ± SEM) in the presence of a Cl⁻ secretory gradient (low Cl⁻ on mucosal surface) and was only partially blocked by the addition of glybenclamide (change of 6.9 ± 1.8 μA/cm², **p< 0.05 vs. apical quinine, n=16, ± SEM). The effect of quinine applied to the basolateral surface was less pronounced than apical administration (**p<0.05 vs. apical quinine, n=16, ± SEM). Bumetanide applied to the basolateral surface several minutes after quinine had no effect compared to apical quinine alone. The change attributable to quinine decreased significantly upon omission of the chloride gradient (‡ p <0.001 vs. apical quinine in Cl⁻ secretory gradient, n=16, ± SEM). In a reversed chloride gradient, the direction of apparent Cl⁻ movement was reversed (‡‡ p <0.001 vs. apical quinine in Cl⁻ secretory gradient, n = 12, ±SEM). B: In Calu-3 monolayers, apical quinine (1 mg/mL) added in a Cl⁻ secretory gradient caused a decrease in airway epithelial resistance (*p= 0.004, n=16, ±SEM) in Calu-3 cells. C: Quinine (1 mg/mL) had no effect on gross epithelial morphology in Calu-3 monolayers studied for localization of tight junctions (Z0-1, green) and CFTR (red). D: Effects of quinine (1 mg/mL) on Ussing chamber recording and transepithelial electrical resistance (E) in primary airway surface epithelial cell monolayers (n = 8 filters tested, ±SEM).

Figure 2. Quinine increases Cl⁻ permeability in airway epithelia through a paracellular pathway associated with decreased epithelial resistance

A: In Calu-3 monolayers, apical quinine (1 mg/mL) increased the monolayer permeability (19.4 ± 3.4 μA/cm², as also shown in Figure 1A,* p < 0.001 compared with untreated controls, n = 16, ± SEM) in the presence of a Cl⁻ secretory gradient (low Cl⁻ on mucosal surface) and was only partially blocked by the addition of glybenclamide (change of 6.9 ± 1.8 μA/cm², **p< 0.05 vs. apical quinine, n=16, ± SEM). The effect of quinine applied to the basolateral surface was less pronounced than apical administration (**p<0.05 vs. apical quinine, n=16, ± SEM). Bumetanide applied to the basolateral surface several minutes after quinine had no effect compared to apical quinine alone. The change attributable to quinine decreased significantly upon omission of the chloride gradient (‡ p <0.001 vs. apical quinine in Cl⁻ secretory gradient, n=16, ± SEM). In a reversed chloride gradient, the direction of apparent Cl⁻ movement was reversed (‡‡ p <0.001 vs. apical quinine in Cl⁻ secretory gradient, n = 12, ±SEM). B: In Calu-3 monolayers, apical quinine (1 mg/mL) added in a Cl⁻ secretory gradient caused a decrease in airway epithelial resistance (*p= 0.004, n=16, ±SEM) in Calu-3 cells. C: Quinine (1 mg/mL) had no effect on gross epithelial morphology in Calu-3 monolayers studied for localization of tight junctions (Z0-1, green) and CFTR (red). D: Effects of quinine (1 mg/mL) on Ussing chamber recording and transepithelial electrical resistance (E) in primary airway surface epithelial cell monolayers (n = 8 filters tested, ±SEM).

Figure 2. Quinine increases Cl⁻ permeability in airway epithelia through a paracellular pathway associated with decreased epithelial resistance

A: In Calu-3 monolayers, apical quinine (1 mg/mL) increased the monolayer permeability (19.4 ± 3.4 μA/cm², as also shown in Figure 1A,* p < 0.001 compared with untreated controls, n = 16, ± SEM) in the presence of a Cl⁻ secretory gradient (low Cl⁻ on mucosal surface) and was only partially blocked by the addition of glybenclamide (change of 6.9 ± 1.8 μA/cm², **p< 0.05 vs. apical quinine, n=16, ± SEM). The effect of quinine applied to the basolateral surface was less pronounced than apical administration (**p<0.05 vs. apical quinine, n=16, ± SEM). Bumetanide applied to the basolateral surface several minutes after quinine had no effect compared to apical quinine alone. The change attributable to quinine decreased significantly upon omission of the chloride gradient (‡ p <0.001 vs. apical quinine in Cl⁻ secretory gradient, n=16, ± SEM). In a reversed chloride gradient, the direction of apparent Cl⁻ movement was reversed (‡‡ p <0.001 vs. apical quinine in Cl⁻ secretory gradient, n = 12, ±SEM). B: In Calu-3 monolayers, apical quinine (1 mg/mL) added in a Cl⁻ secretory gradient caused a decrease in airway epithelial resistance (*p= 0.004, n=16, ±SEM) in Calu-3 cells. C: Quinine (1 mg/mL) had no effect on gross epithelial morphology in Calu-3 monolayers studied for localization of tight junctions (Z0-1, green) and CFTR (red). D: Effects of quinine (1 mg/mL) on Ussing chamber recording and transepithelial electrical resistance (E) in primary airway surface epithelial cell monolayers (n = 8 filters tested, ±SEM).

Figure 3. Influence of 1 mg/ml quinine on transepithelial chloride secretion activated by forskolin

A: Representative I_SC tracing. Calu-3 cells initially studied in Ringers solution followed by 1) mucosal low- Cl⁻ solution, 2) addition of 20 μM forskolin, 3) addition of 1mg/mL quinine, and 4)addition of 200 μM glybenclamide. B: After maximal activation of CFTR, addition of high-dose quinine (1 mg/ml) modestly increased apparent I_SC in a chloride secretory gradient (17.0 ± 3.9 μA/cm², *p<0.005 compared to forskolin alone, n=18, ±SEM) and was partially inhibited by glybenclamide (*p<0.005, n=18, ±SEM). The apparent reversal of I_SC due to quinine in a reversed chloride gradient is attributable to increased paracellular permeability (*p<0.001, n=14, ±SEM).

Figure 4. Quinine blocks CFTR dependent Cl⁻ secretion at concentrations that do not alter airway epithelial resistance

A: In the presence of a Cl⁻ gradient, apical quinine added at 100 μg/mL led to a modest change (7.4 μA/cm²) in I_SC. After activation of CFTR with forskolin (20 μM), substantial reduction in short-circuit current was seen with apical quinine (100 μg/mL, *p<0.05, n=12, ±SEM). Addition of glybenclamide (Glyb, 200 μM) further reduced I_SC(**p<0.005, n=12, ±SEM). In a reversed Cl⁻ gradient, low dose quinine by itself had minimal effect (−5.8 μA/cm², n=13, ±SEM), indicating functionally intact junctional complexes. B: No significant change in monolayer resistance was observed with 100 μg/mL quinine (n=12, ±SEM).