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The effect of sildenafil on chloride secretion in normal and transgenic murine cystic fibrosis colonic epithelia
The effect of sildenafil on electrogenic chloride secretion, in wild type (WT) and cystic fibrosis (CF) transgenic mice colonic epithelia, was investigated using the voltage-clamp technique to measure short circuit current (SCC). Sildenafil, more commonly known as Viagra® (Pfizer UK) , is a PDE5 specific inhibitor and is a clinically approved drug used to treat erectile dysfunction. ΔF508-CFTR transgenic mice contain a deletion of a triplet codon at position 508 in the cystic fibrosis transmembrane regulator (CFTR) gene, resulting in a missing phenylalanine – the most common mutation found in human CF patients. Maturation and processing of Δ F508-CFTR is defective which leads to expression of only very little, but functional, CFTR in the cell membrane. In WT colonic epithelia, sildenafil caused a dose-dependent increase in SCC with an EC50 of 1.7x10-5M. Inhibition of the current by frusemide and the failure of colons to respond in CF-ΔF508 and the transgenic null (knockout) murine model for CF indicate that the increase in SCC caused by sildenafil was due to chloride secretion. The adenylate cyclase promoter, forskolin, also increased electrogenic chloride secretion in WT but not CF colonic epithelia but by a far greater degree than sildenafil, with an EC 50 of 3.1x10 -7M. Forskolin (0.3µM) had the ability to potentiate the dose response produced by sildenafil in WT (but not ΔF508-CFTR) by five-fold. When the reverse situation was investigated sildenafil (100µM) also reduced by five-fold the EC50 for forskolin, again without effect on CF tissues. Six hour incubation with 100µM sildenafil in CF-ΔF508 colons resulted in a significant (p<0.05) increase in the level of forskolin (10µM) -stimulated chloride secretion when compared with tissues without incubation. The mean value observed in non-incubated tissues was -3.60 ± 8.91µAcm-2 (n=4) compared to 20.28 ± 4.17µAcm-2 (n=7) in those exposed to sildenafil for 6 hours. This latter value corresponds to ~20% of the response to the same concentration of forskolin in WT epithelia. No potentiation in response was observed in WT colonic preparations under the same conditions. Our results confirm previous findings in cultured epithelial cell lines (Cobb et al 2003) and in human nasal epithelial cells (Dormer et al, 2005) that sildenafil works to increase chloride secretion by two methods: increasing intracellular levels of cyclic GMP by inhibiting its breakdown and also by increasing levels of apical membrane translocation of ΔF508-CFTR. The data provides further encouragement for future testing of sildenafil in patients with CF.
Cobb B.R. et al (2003) Am J Respir Cell Mol Biol , 29:410-8. |
