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Vascular smooth muscle PDE5 is much more sensitive to N-alkylated zaprinast than platelet PDE5
© Kameni-Tcheudji et al; licensee BioMed Central Ltd. 2007
Published: 25 July 2007
Cyclic nucleotide phosphodiesterases (PDEs) play a key role in signal transduction downstream RCPGs by hydrolyzing specifically cyclic nucleotides. PDE is a superfamily of enzymes presently constituted of 11 gene families . Among them, the PDE5 family is constituted by an unique gene with three different splice variants , and is characterized by its specificity for cGMP hydrolysis, the presence of cGMP-binding sites and its insensitivity to calcium-camoldulin. We have characterized PDE5 in vascular smooth muscle from human, bovine and rat aorta, and showed that zaprinast specifically inhibits PDE5 . The specific inhibition of vascular PDE5 by zaprinast (1 μM) induces relaxation of rat aorta along with an increase in cGMP level . Furthermore, we showed that PDE5 in platelets participates in controling aggregation . The aim of the present study was to compare peptidic sequences, biochemical properties and pharmacological sensitivities of vascular PDE5 and platelet PDE5.
Materials and methods
Cytosolic fractions of smooth muscle from bovine aorta and from human platelet were submitted to DEAE-sepharose chromatography and subsequently to affinity chromatography on ECH-Sepharose 4B resin coupled with NH2-zaprinast. Fractions containing PDE5 were submitted to Western blot and to gel electrophoresis. PDE5 bands were excised from gel and submitted to gel-digestion in order to perform MALDI-TOF analysis. PDE activity was determined by a radioenzymatic assay using 3H-cGMP as a tracer ; Km and Vmax determinations as well as Ki were determined on purified PDE5s. N-alkylated zaprinast was synthetized by L Lebeau using as a scaffold NH2-zaprinast.
MALDI-TOF analysis obtained for trypsic peptides from the 90-kDa Coomassie band of vascular PDE5 (Figure 1A, lane 2) allowed to identify, after Mascot Search, this vascular protein as belonging to PDE5 family when compared with bovine lung PDE5, with a significant score of 126 (significant score being > 60). A similar analysis performed on the 85-kDa band from platelet showed a 32% overlap when compared with the bovine lung PDE5 sequence. Furthermore, a score of 148 was obtained for matching platelet PDE5 sequence with human PDE5A2 sequence. Altogether, these data show that vascular smooth muscle PDE5 and human platelet PDE5 are different proteins.
Biochemical and pharmacological comparisons of vascular and platelet PDE5s
0.44 ± 0.02
0.31 ± 0.04
0.90 ± 0.04
0.71 ± 0.03
K i values (nM)
Our results show that vascular PDE5 and platelet PDE5 are different proteins which can be discriminate by N-alkylated-zaprinast and suggest that they might represent different PDE5 variants which can be selectively inhibited. These data open new therapeutic opportunities for using PDE5 inhibitor treatment in patients treated with antiaggregant.
We are grateful to Dr Jackie Corbin (Vandebilt University, USA) for providing us with the bovine lung PDE5 antibody and we acknowledge the skillful technical help of Hélène Justiniano.
- Lugnier C: Cyclic Nucleotide Phosphodiesterase (PDE) superfamily: A New Target for the Development of Specific Therapeutic Agents. Pharmacol & Ther. 2006, 109: 366-398. 10.1016/j.pharmthera.2005.07.003.View ArticleGoogle Scholar
- Lin CS, Lau A, Tu R, Lue TF: Expression of three isoforms of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in human penile cavernosum. Biochem Biophys Res Commun. 2000, 268: 628-635. 10.1006/bbrc.2000.2187.View ArticlePubMedGoogle Scholar
- Lugnier C, Schoeffter P, Le Bec A, Strouthou E, Stoclet JC: Selective inhibition of cyclic nucleotide phosphodiesterases of human, bovine and rat aorta. Biochem Pharmacol. 1986, 35: 1743-1751. 10.1016/0006-2952(86)90333-3.View ArticlePubMedGoogle Scholar
- Schoeffter P, Lugnier C, Demesy-Waeldele F, Stoclet JC: Role of cyclic AMP- and cyclic GMP-phosphodiesterases in the control of cyclic nucleotide levels and smooth muscle tone in rat isolated aorta: A study with selective inhibitors. Biochem Pharmacol. 1987, 36: 3965-3972. 10.1016/0006-2952(87)90465-5.View ArticlePubMedGoogle Scholar
- Lugnier C, Stierle A, Beretz A, Schoeffter P, Le Bec A, Wermuth CG, Cazenave JP, Stoclet JC: Tissue and substrate specificity of inhibition by alkoxy-aryl-lactams of platelet and arterial smooth muscle cyclic nucleotide phosphodiesterases relationship to pharmacological activity. Biochem Biophys Res Commun. 1983, 113: 954-959. 10.1016/0006-291X(83)91091-4.View ArticlePubMedGoogle Scholar
- Keravis T, Thaseldar-Roumie R, Lugnier C: Assessment of phosphodiesterase isozyme contribution in cell and tissue extracts. Meth Mol Biol. 2005, 307: 63-74.Google Scholar
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