High lung PDE5: A strong basis for treating pulmonary hypertension with PDE5 inhibitors,☆☆

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Abstract

[3H]Vardenafil (Levitra) or [3H]tadalafil (Cialis) binding was used to quantify PDE5 in rat lung and heart tissue. Each radioligand bound to purified recombinant phosphodiesterase-5 (PDE5) or to PDE5 in crude extracts with strong affinity, high specificity, slow dissociation, and good stoichiometry. PDE5, the only 3H inhibitor-binding protein detected in extracts, was 15 times higher in lung than in heart extracts, and the level measured by PDE5 catalytic activity agreed with that determined by 3H inhibitor binding. High level of PDE5 in lung approximated that in penile corpus cavernosum, the tissue targeted by PDE5 inhibitors. PDE5 was the predominant cGMP-PDE in lung, and on a molar basis was five times higher than cGMP-dependent protein kinase (PKG), which phosphorylates PDE5 in vivo. The PDE5 level was one-half that of PKG in heart. Thus, abundance of PDE5 in lung vascular smooth muscle provides a strong molecular basis for PDE5 inhibitor treatment of pulmonary hypertension.

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Materials and methods

Materials. [3H]cGMP and [3H]cAMP, were purchased from Amersham Biosciences (Piscataway, NJ). Sephadex G-25, and DEAE–Sephacel were from Pfizer. Histone type II-AS, histone type VIII-S, 3-isobutyl-1-methylxanthine (IBMX), 8-chlorophenylthio-cGMP (8-CPT-cGMP), Crotalus atrox snake venom, cilostamide, rolipram, cAMP, and cGMP were from Sigma Chemical (St. Louis, MO). PKG-selective heptapeptide (RKRSRAE), PKA-selective heptapeptide (LRRASLG, Kemptide), and PKItide (protein kinase inhibitor peptide,

3H inhibitor binding in crude extracts of rat lung and heart

[3H]Vardenafil and [3H]tadalafil were shown to bind with high affinity, stoichiometry (nearly 1 mol inhibitor per PDE5 subunit), and specificity for the catalytic domain of purified, recombinant PDE5 [18]. Thus, it seemed logical that these 3H inhibitor-binding assays could be used to estimate the levels of PDE5 in crude fractions of tissue extracts. Using the supernatant from rat heart, the time course and volume-dependence of the sample for binding of [3H]vardenafil at 4 °C are shown in Fig. 1.

Discussion

The traditional approach of using PDE catalytic activity to quantify a particular PDE is oftentimes inadequate. The contribution of other PDEs to the observed PDE activity can be substantial even after optimization of assay conditions (substrate, ±PDE inhibitors, etc.). This is much less of a concern using 3H inhibitor-binding activity, which has not yet revealed the presence of detectable 3H inhibitor binding proteins other than PDE5 in a limited number of tissue extracts. The binding assay

Acknowledgments

We thank Bayer for providing vardenafil and [3H]vardenafil, and ICOS Corporation for providing tadalafil. We are grateful to E. Bronson Ingram Cancer Center and Diabetes Center of Vanderbilt University.

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    This work was supported by NIH DK58277, DK40029, and research grants from Bayer Pharmaceuticals and ICOS Corporation.

    ☆☆

    Abbreviations: PDE, 3′,5′-cyclic nucleotide phosphodiesterase; PDE5, cGMP-binding cGMP-specific PDE; KPM, 10 mM potassium phosphate, pH 6.8, and 25 mM β-mercaptoethanol; IBMX, 3-isobutyl-1-methylxanthine; PKG, cGMP-dependent protein kinase; PKA, cAMP-dependent protein kinase; 8-CPT-cGMP, 8-chlorophenylthio-cGMP; PKI, protein kinase inhibitor peptide 5–24.

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