In addition to its cGMP-selective catalytic site, cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains two allosteric cGMP-binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem. 265, 14971±14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture [cGMP-dependent protein kinase (PKG) or catalytic subunit of cAMP-dependent protein kinase (PKA), MgATP, cGMP, 3-isobutyl-1-methylxanthine], shown earlier to produce Ser92 phosphorylation, caused a 50±70% increase in enzyme activity and also increased the affinity of cGMP binding to the allosteric cGMP-binding sites. Both effects were associated with increases in its phosphate content up to 0.6 mol per PDE5 subunit. Omission of any one of the preincubation components caused loss of stimulation of catalytic activity. Addition of the phosphorylation reaction mixture to a crude bovine lung extract, which contains PDE5, also produced a significant increase in cGMP PDE catalytic activity. The increase in recombinant PDE5 catalytic activity brought about by phosphorylation was time-dependent and was obtained with 0.2±0.5 mm PKG subunit, which is approximately the cellular level of this enzyme in vascular smooth muscle. Significantly greater stimulation was observed using cGMP substrate concentrations below the K m value for PDE5, although stimulation was also seen at high cGMP concentrations. Considerably higher concentration of the catalytic subunit of PKA than of PKG was required for activation. There was no detectable difference between phosphorylated and unphosphorylated PDE5 in median inhibitory concentration for the PDE5 inhibitors, sildenafil, or zaprinast 3-isobutyl-1-methylxanthine. Phosphorylation reduced the cGMP concentration required for half-maximum binding to the allosteric cGMP-binding sites from 0.13 to 0.03 mm. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative-feedback regulation of cGMP levels is discussed.

Sildenafil, tadalafil, and vardenafil each competitively inhibit cGMP hydrolysis by phosphodiesterase-5 (PDE5), thereby fostering cGMP accumulation and relaxation of vascular smooth muscle. Biochemical potencies (affinities) of these compounds for PDE5 determined by IC 50 , K D (isotherm), K D (dissociation rate), and K D ( 1 ⁄2 EC 50 ), respectively, were the following: sildenafil (3.7 Ϯ 1.4, 4.8 Ϯ 0.80, 3.7 Ϯ 0.29, and 11.7 Ϯ 0.70 nM), tadalafil (1.8 Ϯ 0.40, 2.4 Ϯ 0.60, 1.9 Ϯ 0.37, and 2.7 Ϯ 0.25 nM); and vardenafil (0.091 Ϯ 0.031, 0.38 Ϯ 0.07, 0.27 Ϯ 0.01, and 0.42 Ϯ 0.10 nM). Thus, absolute potency values were similar for each inhibitor, and relative potencies were vardenafil Ͼ Ͼ tadalafil Ͼ sildenafil. Binding of each 3 H inhibitor to PDE5 was specific as determined by effects of unlabeled compounds.

No abstract

The physiological role of phosphodiesterase (PDE)11 is unknown and its biochemical characteristics are poorly understood. We have expressed human His-tagged PDE11A4 and purified the enzyme to apparent homogeneity. PDE11A4 displays K m values of 0.97 lM for cGMP and 2.4 lM for cAMP, and maximal velocities were 4-to 10-fold higher for cAMP than for cGMP. Given the homology between PDE11 and PDE5, we have compared the biochemical potencies of tadalafil (Cialist, Lilly-ICOS), vardenafil (Levitrat, Bayer-GSK), and sildenafil (Viagrat, Pfizer Inc.) for PDE11A4 and PDE5A1. PDE5A1/PDE11A4 selectivities are 40-, 9300-, and 1000-fold for tadalafil, vardenafil, and sildenafil, respectively. This suggests that none of these three compounds is likely to crossreact with PDE11A4 in patients.

Sildenafil (Viagra) potentiates penile erection by acting as a nonhydrolyzable analog of cGMP and competing with this