Phosphodiesterase-5 isoforms: differential cyclic guanyl monophosphate binding and cyclic guanyl monophosphate catalytic activities, and inhibitory effects of sildenafil and vardenafil.

PURPOSE

We determined the differential cyclic guanyl monophosphate catalytic and cyclic guanyl monophosphate binding activity of phosphodiesterase-5 isoforms and the inhibitory effects of sildenafil (Pfizer, New York, New York) and vardenafil (Bayer Pharmaceutical Research, Wuppertal, Germany).

MATERIALS AND METHODS

Coding sequences of the human phosphodiesterase-5 isoforms A1, A2 and A3 were cloned into pBlueBac4.5/V5-His (Invitrogen, Carlsbad, California), which allows the tagging of histidines at the carboxyl terminal of the expressed protein. Isoforms were expressed with the Bac-N-Blue baculoviral system and purified with the ProBond system. Expression clones were identified by polymerase chain reaction using vector and phosphodiesterase-5 specific primers. Purified proteins were verified by Western blotting. Purified proteins were analyzed for cyclic guanyl monophosphate catalytic and cyclic guanyl monophosphate binding activity, and used to determine the differential potencies of the phosphodiesterase-5 selective inhibitors sildenafil and vardenafil.

RESULTS

Cloning and expression of phosphodiesterase-5A1 to A3 isoforms in the baculoviral system resulted in the isolation of purified isoform proteins. Mean cyclic guanyl monophosphate catalytic activity (K(m)) +/- SD was 4.76 +/- 0.37, 4.52 +/- 0.09 and 11.39 +/- 0.22 microM for A1 to A3, respectively. Mean cyclic guanyl monophosphate binding activity (K(d)) was 3.24 +/- 0.47, 1.95 +/- 0.60 and 1.70 +/- 0.47 microM for A1 to A3, respectively. Mean IC(50) of sildenafil against phosphodiesterase-5A1 to A3 was 1.20 +/- 0.34, 2.83 +/- 0.56 and 2.28 +/- 0.38 nM, respectively. Mean IC(50) of vardenafil against phosphodiesterase-5A1 to A3 was 0.41 +/- 0.15, 0.23 +/- 0.08 and 0.45 +/- 0.06 nM, respectively.

CONCLUSIONS

Phosphodiesterase-5A1 and A2 had similar K(m) values, while phosphodiesterase-5A3 had a much higher K(m) and, thus, lower cyclic guanyl monophosphate catalytic activity. Phosphodiesterase-5A2 and A3 had similar K(d) values, while phosphodiesterase-5A1 had higher K(d) and, thus, lower cyclic guanyl monophosphate binding activity. Vardenafil was more potent (3 to 12-fold) than sildenafil for inhibiting the catalytic activity of all 3 human phosphodiesterase-5 isoforms with phosphodiesterase-5A2 showing the highest differentiation (12-fold).