Biochemical and Biophysical Research Communications
High lung PDE5: A strong basis for treating pulmonary hypertension with PDE5 inhibitors☆,☆☆
Section snippets
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.
References (30)
- et al.
Lancet
(2002) - et al.
J. Am. Coll. Cardiol.
(2004) - et al.
J. Biol. Chem.
(1980) - et al.
J. Biol. Chem.
(1990) - et al.
J. Biol. Chem.
(1999) - et al.
J. Biol. Chem.
(1982) - et al.
Cell Signal.
(2004) - et al.
J. Biol. Chem.
(1990) - et al.
Eur. J. Biochem.
(2001) - et al.
Circulation
(2002)
Circulation
Circulation
Proc. Natl. Acad. Sci. USA
Prog. Nucleic Acid Res. Mol. Biol.
J. Appl. Physiol.
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This work was supported by NIH DK58277, DK40029, and research grants from Bayer Pharmaceuticals and ICOS Corporation.
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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.