Cyclic GMP Phosphodiesterase-5: Target of Sildenafil*

The advent of the medication, sildenafil, for treatment of male impotence has attracted widespread attention. This agent potently inhibits a cGMP-binding cGMP-specific phosphodiesterase (PDE5). PDE5 is particularly abundant in smooth muscle, which is enriched in other components of the cGMP signaling cascade. The characteristics of PDE5, its relationship to other PDEs, its role in cGMP signaling, and its involvement in the efficacious action of sildenafil on corpus cavernosum and vascular smooth muscle resulting in penile erection are the subjects of this review. Cyclic GMP has emerged recently as a principal focus in signal transduction. Much of this attention has derived from the fact that most of the non-lytic physiological effects of nitric oxide (Fig. 1) and all of the characterized effects of natriuretic peptides and guanylins are mediated by cGMP. In addition to the classical regulatory roles ascribed to cGMP such as stimulation of smooth muscle relaxation, neutrophil degranulation, inhibition of platelet aggregation, and initiation of visual signal transduction, numerous other physiological roles have recently been uncovered (1–10). Intracellular receptors for cGMP include cGMP-dependent protein kinases (PKG), cyclic nucleotide-gated channels, and cGMP-binding PDEs; cGMP may also cross-activate cAMP pathways by binding to cAMP-binding sites on cAMP receptors such as cAMP-dependent protein kinases (PKA) (11). Tissue cGMP levels are determined by a balance between the activities of the guanylyl cyclases that catalyze formation of cGMP from GTP and the cyclic nucleotide PDEs that catalyze the breakdown of cGMP (Fig. 1). The combination of a stimulator of guanylyl cyclase and a cGMP PDE inhibitor such as sildenafil produces synergistic enhancement of tissue cGMP levels (12). PDEs were first detected by Sutherland and co-workers (13, 14). The superfamily of PDEs is subdivided into two major classes, class I and class II (15), which have no recognizable sequence similarity. Class I includes all known mammalian PDEs and is comprised of at least 10 identified families that are products of separate genes (16–26). Some PDEs are highly specific for hydrolysis of cAMP (PDE4, PDE7, PDE8), some are highly cGMP-specific (PDE5, PDE6, PDE9), and some have mixed specificity (PDE1, PDE2, PDE3, PDE10). All of the characterized mammalian PDEs are dimeric, but the importance of the dimeric structure for function in each of the PDEs is unknown. Each PDE has a conserved catalytic domain of ;270 amino acids with a high degree of conservation (25–30%) of amino acid sequence among PDE families, which is located carboxyl-terminal to its regulatory domain. Activators of certain PDEs appear to relieve the influence of autoinhibitory domains located within the enzyme structures (27, 28). PDEs cleave the cyclic nucleotide phosphodiester bond between the phosphorus and oxygen atoms at the 39-position with inversion of configuration at the phosphorus atom (29, 30). This apparently results from an in-line nucleophilic attack by the OH of ionized H2O. It has been proposed that metals bound in the conserved metal binding motifs within PDEs facilitate the production of the attacking OH (31). The kinetic properties of catalysis are consistent with a random order mechanism with respect to cyclic nucleotide and the divalent cation(s) that are required for catalysis (32). The catalytic domains of all known mammalian PDEs contain two sequences (HX3HXn(E/D)) arranged in tandem, each of which resembles the single Zn-binding site of metalloendoproteases such as thermolysin (31). PDE5 specifically binds Zn, and the catalytic activities of PDE4, PDE5, and PDE6 are supported by submicromolar concentrations of Zn (31, 33). Whether each of the Zn binding motifs binds Zn independently or whether the two motifs interact to form a novel Zn-binding site is not known. The catalytic mechanism for cleaving phosphodiester bonds of cyclic nucleotides by PDEs may be similar to that of certain proteases for cleaving the amide ester of peptides, but the presence of two Zn motifs arranged in tandem in PDEs is unprecedented. The group of Sutherland and Rall (34), in the late 1950s, was the first to realize that at least part of the mechanism(s) whereby caffeine enhanced the effect of glucagon, a stimulator of adenylyl cyclase, on cAMP accumulation and glycogenolysis in liver involved inhibition of cAMP PDE activity. Since that time chemists have synthesized thousands of PDE inhibitors, including the widely used 3-isobutyl-1-methylxanthine (IBMX). Many of these compounds, as well as caffeine, are non-selective and inhibit many of the PDE families. One important advance in PDE research has been the discovery/design of family-specific inhibitors such as the PDE4 inhibitor rolipram and the PDE5 inhibitor sildenafil. Precise modulation of PDE function in cells is critical for maintaining cyclic nucleotide levels within a narrow rate-limiting range of concentrations. Increases in cGMP of 2–4-fold above the basal level will usually produce a maximum physiological response. There are three general schemes by which PDEs are regulated: (a) regulation by substrate availability, such as by stimulation of PDE activity by mass action after elevation of cyclic nucleotide levels or by alteration in the rate of hydrolysis of one cyclic nucleotide because of competition by another, which can occur with any of the dual specificity PDEs (e.g. PDE1, PDE2, PDE3); (b) regulation by extracellular signals that alter intracellular signaling (e.g. phosphorylation events, Ca, phosphatidic acid, inositol phosphates, protein-protein interactions, etc.) resulting, for example, in stimulation of PDE3 activity by insulin (18), stimulation of PDE6 activity by photons through the transducin system (35), which alters PDE6 interaction with this enzyme, or stimulation of PDE1 activity by increased interaction with Ca/calmodulin; (c) feedback regulation, such as by phosphorylation of PDE1, PDE3, or PDE4 catalyzed by PKA after cAMP elevation (17, 18, 36, 37), by allosteric cGMP binding to PDE2 to promote breakdown of cAMP or cGMP after cGMP elevation, or by modulation of PDE protein levels, such as the desensitization that occurs by increased concentrations of PDE3 or PDE4 following chronic exposure of cells to cAMP-elevating agents (17, 38) or by developmentally related changes in PDE5 content. Other factors that could influence any of the three schemes outlined above are cellular compartmentalization of PDEs (19) effected by covalent modifications such as prenylation or by specific targeting sequences in the PDE primary structure and perhaps translocation of PDEs between compartments within a cell. Within the PDE superfamily, four (PDE2, PDE5, PDE6, and PDE10) of the 10 families contain highly cGMP-specific allosteric (non-catalytic) cGMP-binding sites in addition to a catalytic site of varying substrate specificity. Each of the monomers of these di* This minireview will be reprinted in the 1999 Minireview Compendium, which will be available in December, 1999. This work was supported by National Institutes of Health Grants GM41269 and DK40029 and American Heart Association Southeast Affiliate. ‡ To whom correspondence and reprint requests should be addressed: Dept. of Molecular Physiology and Biophysics, 702 Light Hall, Vanderbilt University School of Medicine, 21st and Garland Aves., Nashville, TN 372320615. Tel.: 615-322-4384; Fax: 615-343-3794; E-mail: jackie.corbin@ mcmail.vanderbilt.edu. 1 Tradename VIAGRATM. 2 The abbreviations used are: PDE, 39:59-cyclic nucleotide phosphodiesterase; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; IBMX, 3-isobutyl-1-methylxanthine. 3 J. A. Beavo and K. Loughney, personal communication. 4 S. Francis, unpublished results. Minireview THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 20, Issue of May 14, pp. 13729–13732, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.