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Phosphodiesterase 5 Inhibitors and Cystic Fibrosis

Correcting Chloride Channel Dysfunction

University of Missouri
Columbia, Missouri

Fueled by bench studies demonstrating the therapeutic possibilities, a growing effort in cystic fibrosis (CF) research has concentrated on searching for pharmacological compounds that improve the biogenesis and function of the protein product of the major mutation in the CF transmembrane conductance regulator gene, F508 CFTR. Normal CFTR is the principal cyclic nucleotide-activated channel responsible for chloride and bicarbonate secretion across epithelia, a role that provides hydration of mucus, enzymes, and antimicrobial peptides in the airways and other epithelia. More than 90% of all patients with CF carry at least one F508 CFTR allele (1). Thus, improving the activity of F508 CFTR will benefit most of the CF population. However, the task is daunting. Although F508 CFTR is translated, processing of the protein is blocked within the endoplasmic reticulum and directed toward degradation instead of plasma membrane insertion (2). Fortunately, the degradation pathway can be circumvented. High-throughput screening centers are searching for pharmaceutically useful molecules ("correctors") that can facilitate F508 CFTR trafficking (3, 4). However, addressing F508 CFTR trafficking will not completely correct the epithelia alterations associated with CF since F508 CFTR has reduced channel activity (5). Thus, paralleling the search for "correctors" is a search for pharmacological agents to improve F508 CFTR channel function.

One strategy to improve F508 CFTR channel function is pharmacological inhibition of phosphodiesterases (PDEs), the enzymes responsible for inactivating intracellular cyclic nucleotides. Initial efforts to maximize cAMP using xanthine-based inhibitors of PDEs 1–4 demonstrated improved F508 CFTR function in mouse models, expression systems, or cultured cells. However, testing of these inhibitors in human F508 CFTR airway epithelium did not consistently meet with success (6, 7). Less attention was given to inhibiting cyclic guanosine monophosphate (cGMP) degradation, which principally involves the activity of PDE5, an enzyme with high specificity for cGMP.

Although early studies showed that increased cGMP stimulates CFTR activity, the introduction of high-potency PDE5 inhibitors, such as sildenafil, the active ingredient in Viagra used for treating erectile dysfunction in males, enabled a closer examination of cGMP metabolism and CFTR activity. Sildenafil was first shown to improve mucin secretion in a CF cell model (8) and, subsequently, found to stimulate normal CFTR activity in airway cell lines (9). Two reports indicate that very high concentrations (300 x therapeutic levels) of sildenafil increases plasma membrane localization of F508 CFTR (4, 10). More recently, biochemical studies show that therapeutic levels of sildenafil increase cGMP inhibition of intracellular epithelial Na⁺ channel (ENaC) activity and improve trans-Golgi network function in CF airway cells (11). The latter study also provided preliminary evidence that sildenafil treatment activated endogenous F508 mCFTR in the nasal epithelium of F508 CFTR mutant mice.

In this issue of the Journal (pp. 506–515), Lubamba and colleagues provide preclinical evidence that PDE5 inhibitors can restore anion secretion to airway epithelium of F508 CFTR mutant mice in vivo (12). Measuring nasal mucosal potential difference, near normal levels of chloride conductance were achieved within 1 hour after treatment with a clinically relevant dose of sildenafil. Control experiments show that sildenafil does not increase the airway chloride conductance of normal mice (although it potentiates cAMP stimulation), and does not have a stimulatory effect on airway epithelia of CFTR knockout mice, thus demonstrating its specificity for F508 mCFTR. Interestingly, the positive effect of a single dose of sildenafil was not evident after 24 hours, whereas treatment with vardenafil (Levitra), an analog with higher potency and slower elimination, sustained the chloride conductance for 24 hours. In contrast, treatment with PDE5 inhibitors did not decrease high rates of Na⁺ absorption across the F508 CFTR mouse airways. Elevated Na⁺ absorption resulting from abnormal ENaC activity is also characteristic of human CF airways and contributes to mucus dehydration (13). This pathologic feature reflects loss of a CFTR regulatory role. Thus, the present study indicates that this aspect of the disease may require therapeutic considerations beyond improving chloride channel function.

It is important to point out that sildenafil correction of F508 mCFTR chloride conductance in Lubamba and colleagues' study likely results from the activation of membrane-resident F508 mCFTR rather than correction of F508 mCFTR processing. The clue comes from the type of F508 CFTR mouse model. Three F508 CFTR mouse models have been developed by gene targeting (14). Two of the models, cftr^tm1Kth and cftr^tm2Cam, were created by "replacement" gene targeting, which has the attendant consequence of suppressing CFTR transcription by 70–85%. These mice are essentially mCFTR knockouts that show a severe deficit of transepithelial anion secretion and high mortality. In contrast, the cftr^tm1Eur mouse model used in the present study was generated by "hit and run" targeting that preserves intronic structure of the gene. These mice have normal mRNA expression of F508 mCFTR and, since the mouse protein processes better than the human mutant (15), a significant fraction of the F508 mCFTR escapes quality control for insertion in the plasma membrane. The cftr^tm1Eur mice have approximately 33% of normal cAMP-stimulated anion secretion and normal survival. Thus, membrane-resident F508 mCFTR is available for activation by sildenafil. The authors of the study have been careful to avoid the interpretation that the effects of sildenafil are due to F508 mCFTR processing correction. Because both compounds stimulate F508 mCFTR activity within 1 hour, interpretation leans toward pharmacological activation of plasma membrane F508 mCFTR.

The search will clearly continue for correctors of F508 CFTR processing because mutant channels must be plasma membrane resident before channel activity can be restored. Even with restoration of channel activity, the task will not be complete because F508 CFTR is also less stable in the plasma membrane than its normal counterpart (16). Nonetheless, the discovery that clinically available PDE5 inhibitors activate anion secretion and perhaps improve processing of F508 CFTR will stimulate future research for their use in CF. Thus, the focus will sharpen and resolve will strengthen in the quest to pharmacologically manage the deficiencies of the F508 CFTR protein.

FOOTNOTES

Conflict of Interest Statement: L.L.C. has no financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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