doi:10.1016/j.bbrc.2006.01.127 
Copyright © 2006 Elsevier Inc. All rights reserved.
Novel short chain fatty acids restore chloride secretion in cystic fibrosis
Toan D. Nguyena, 
, 
, Ug-Sung Kima and Susan P. Perrineb
aDivision of Gastroenterology, Department of Medicine, University of Washington and VA Puget Sound Health Care System, Seattle, WA 98108, USA
bCancer Center, Departments of Pediatrics, Medicine, Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA 02118, USA
Received 24 January 2006.
Available online 3 February 2006.
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Abstract
Phenylalanine deletion at position 508 of the cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR), the most common mutation in cystic fibrosis (CF), causes a misfolded protein exhibiting partial chloride conductance and impaired trafficking to the plasma membrane. 4-Phenylbutyrate corrects defective ΔF508-CFTR trafficking in vitro, but is not clinically efficacious. From a panel of short chain fatty acid derivatives, we showed that 2,2-dimethyl-butyrate (ST20) and 
-methylhydrocinnamic acid (ST7), exhibiting high oral bioavailability and sustained plasma levels, correct the ΔF508-CFTR defect. Pre-incubation (
6 h) of CF IB3-1 airway cells with 1 mM ST7 or ST20 restored the ability of 100 μM forskolin to stimulate an 125I− efflux. This efflux was fully inhibited by NPPB, DPC, or glibenclamide, suggesting mediation through CFTR. Partial inhibition by DIDS suggests possible contribution from an additional Cl− channel regulated by CFTR. Thus, ST7 and ST20 offer treatment potential for CF caused by the ΔF508 mutation.
Keywords: Cystic fibrosis; ΔF508-CFTR; 2,2-dimethyl-butyrate; -methylhydrocinnamic acid; Iodide efflux; IB3-1 airway cells; Butyrate derivatives; Intracellular trafficking; Chloride channel; Chloride conductance
Fig. 1. Chemical structures of butyrate (AB), 4-phenyl butyric acid (4-PBA), -methyl hydrocinnamic acid (ST7), and 2,2-dimethyl-butyrate (ST20).
Fig. 2. Effect of SCFAD on forskolin-stimulated 125I− efflux from IB3-1 cells. Confluent monolayers of IB3-1 cells were pretreated for 2 days with 2.5 mM of either 4-PBA (•), AB (□), ST7 (Δ), ST20 (
), or arginine-containing vehicle (control, ○) and the 125I− efflux stimulated by 100 μM forskolin determined as outlined in the Methods. The mean and SEM from three different experiments are shown for each data point. The inset shows the increases in peak efflux rate coefficients (coefficient at 2.25 min minus coefficient at 1 min) which were, respectively, −0.011 ± 0.007/min, 0.051 ± 0.006/min, 0.040 ± 0.011/min, 0.045 ± 0.003/min, and 0.077 ± 0.009/min, for untreated cells and cells treated with 4-PBA, AB, ST7, and ST20 (p < 0.05 for all SCFAD treatments vs. untreated control).
Fig. 3. Dependence of SCFAD effect on duration of pretreatment.Confluent monolayers of IB3-1 cells were pretreated for the indicated lengths of time with 2.5 mM of either AB (A), ST7 (B), or ST20 (C), and the 125I− efflux stimulated by 100 μM forskolin determined as outlined in the Methods. The mean and SEM from three different experiments are shown for each data point.
Fig. 4. Concentration-dependence of SCFAD effect. Confluent monolayers of IB3-1 cells were pretreated for 2 days with either AB (A), ST7 (B), or ST20 (C), at the indicated concentrations, and the 125I− efflux stimulated by 100 μM forskolin determined as outlined in the Methods. The mean and SEM from three different experiments are shown for each data point.
Fig. 5. Inhibition of forskolin-stimulated 125I− efflux following SCFAD treatment by inhibitors of chloride channels. Confluent monolayers of IB3-1 cells were pretreated for 2 days with either 5 mM AB (A), ST7 (B), or ST20 (C), and the 125I− efflux stimulated by 100 μM forskolin in the presence or absence of 500 μM glibenclamide, 500 μM NPPB, 2.5 mM DPC, or 500 μM DIDS, determined as outlined in the Methods. The means and SEM from three different experiments are shown.
Fig. 6. Comparison of ST20 and aminoglycoside treatment on forskolin-stimulated 125I− efflux. Confluent monolayers of IB3-1 cells were pretreated for 2 days with 2.5 mM ST20, 200 μg/ml G418 (A), or 200 μg/ml gentamycin, (B) alone or in combination, and the 125I− efflux subsequently stimulated by 100 μM forskolin determined as outlined in the Methods. The mean and SEM from three different experiments are shown for each data point. The experiments in this figure were generated together but separated into two panels for clarity.
Table 1.
Effects of added SCFAD on the 125I− efflux from IB3-1 cells
Confluent IB3-1 cells were treated with 2.5 mM SCFAD for 2 days and the 125I− efflux stimulated by 100 μM forskolin was determined as described in the Methods. The peak change of efflux rate coefficients was calculated by subtracting the baseline efflux at 1 min (just prior to the addition of forskolin) from the peak efflux at 2.5 min (1.5 min following forskolin). A positive response denotes a robust peak 125I− efflux increase that was statistically different from control (n = 3, p < 0.05 by unpaired two-tailed t test); a borderline response denotes a discernible 125I− increase which did not reach statistical significance; and a negative response denotes no discernible 125I− efflux increase.
a Cellular injury as suggested by the detachment of cells from the filter.
Table 2.
Dependence of SCFAD effect on duration of pre-incubation
IB3-1 cells were pre-incubated with AB, ST7, or ST20, or with arginine-containing vehicle (untreated control) for different time periods and the subsequent 125I− efflux stimulated by 100 μM forskolin was determined, as shown in Fig. 3. The peak changes of efflux rate coefficients, calculated by subtracting the baseline efflux at 1 min from the peak effluxes at 2.5 min (for AB and ST7) and 2.75 min (for ST20), are shown (mean ± SEM). For each SCFAD, the experiments were performed on the same day, using cells grown in parallel; * and † denote statistically significant differences (p < 0.05 by two-tailed t test and one-tailed t test, respectively) with the untreated control. Because the different SCFAD were tested separately, results between different SCFAD in this table are not comparable.
Table 3.
Concentration-dependency of SCFAD correction
IB3-1 cells were pre-incubated with different concentrations of AB, ST7, or ST20 for 2 days and the subsequent 125I− efflux stimulated by 100 μM forskolin determined, as shown in Fig. 4. The peak changes of efflux rate coefficients, calculated by subtracting the baseline efflux at 1 min from the peak effluxes at 2.5 min (for AB), 2.5–3.5 min (for ST7), and 2.75 min (for ST20), are shown (mean ± SEM). For each SCFAD, the experiments were performed on the same day, using cells grown in parallel; because the different SCFAD were tested separately, results between different SCFAD in this table are not comparable. p < 0.05: *vs. control by unpaired two-tailed t test, †vs. control by unpaired one-tailed t test, %vs. 2.5 mM by unpaired two-tailed t test.
a Peak effluxes used occurred at 2.5 min for 1 mM, 3.5 min for 2.5 mM, and 3 min for 5 mM.
b Peak effluxes used occurred at 2.25, 2.5, and 2.75 min.
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