[3H]Sildenafil binding to phosphodiesterase-5 is specific, kinetically heterogeneous, and stimulated by cGMP

Sildenafil ( Viagra) potentiates penile erection by acting as a nonhydrolyzable analog of cGMP and competing with this nucleotide for catalysis by phosphodiesterase-5 (PDE5), but the characteristics of direct binding of radiolabeled sildenafil to PDE5 have not been determined. [H-3] Sildenafil binding to PDE5 was retained when filtered through nitrocellulose or glass-fiber membranes. Binding was inhibited by excess sildenafil, 2-(2-methylpyridin- 4-yl) methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2- yl) methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T-0156), 3-isobutyl-1- methylxanthine, EDTA, or cGMP, but not by cAMP or 5'-GMP. PDE5 was the only [H-3] sildenafil binding protein detected in human lung extract. Using purified recombinant PDE5, [H-3] sildenafil exchange dissociation yielded two components with t(1/2) values of 1 and 14 min and corresponding calculated K-D values of 12 and 0.83 nM, respectively. This implied the existence of two conformers of the PDE5 catalytic site. [H-3] Sildenafil binding isotherm of PDE5 indicated K-D was 8.3 to 13.3 nM, and low cGMP decreased the K-D to 4.8 nM but only slightly increased B-max to a maximum of 0.61 mol/mol-subunit. Results suggest that these effects occur via cGMP binding to the allosteric cGMP binding sites of PDE5. Results imply that by inhibiting PDE5 and thereby increasing cGMP, sildenafil accentuates its own binding affinity for PDE5, which further elevates cGMP. The data also indicate that after physiological elevation, cGMP may directly stimulate the catalytic site by binding to the allosteric cGMP-binding sites of PDE5, thus causing negative feedback on this pathway.