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Research ArticleArticle

[3H]Sildenafil Binding to Phosphodiesterase-5 Is Specific, Kinetically Heterogeneous, and Stimulated by cGMP

Jackie D. Corbin, Mitsi A. Blount, James L. Weeks II, Alfreda Beasley, Karl P. Kuhn, Yew S. J. Ho, Layla F. Saidi, James H. Hurley, Jun Kotera and Sharron H. Francis
Molecular Pharmacology June 2003, 63 (6) 1364-1372; DOI: https://doi.org/10.1124/mol.63.6.1364
Jackie D. Corbin
Department of Molecular Physiology and Biophysics (J.D.C., M.A.B., J.L.W., A.B., J.K., S.H.F.) and Center for Lung Research, Division of Allergy, Pulmonary and Critical Care Medicine (K.P.K.), Vanderbilt University School of Medicine, Nashville, Tennessee; and Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (Y.S.J.H., L.F.S., J.H.H.)
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Mitsi A. Blount
Department of Molecular Physiology and Biophysics (J.D.C., M.A.B., J.L.W., A.B., J.K., S.H.F.) and Center for Lung Research, Division of Allergy, Pulmonary and Critical Care Medicine (K.P.K.), Vanderbilt University School of Medicine, Nashville, Tennessee; and Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (Y.S.J.H., L.F.S., J.H.H.)
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James L. Weeks II
Department of Molecular Physiology and Biophysics (J.D.C., M.A.B., J.L.W., A.B., J.K., S.H.F.) and Center for Lung Research, Division of Allergy, Pulmonary and Critical Care Medicine (K.P.K.), Vanderbilt University School of Medicine, Nashville, Tennessee; and Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (Y.S.J.H., L.F.S., J.H.H.)
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Alfreda Beasley
Department of Molecular Physiology and Biophysics (J.D.C., M.A.B., J.L.W., A.B., J.K., S.H.F.) and Center for Lung Research, Division of Allergy, Pulmonary and Critical Care Medicine (K.P.K.), Vanderbilt University School of Medicine, Nashville, Tennessee; and Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (Y.S.J.H., L.F.S., J.H.H.)
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Karl P. Kuhn
Department of Molecular Physiology and Biophysics (J.D.C., M.A.B., J.L.W., A.B., J.K., S.H.F.) and Center for Lung Research, Division of Allergy, Pulmonary and Critical Care Medicine (K.P.K.), Vanderbilt University School of Medicine, Nashville, Tennessee; and Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (Y.S.J.H., L.F.S., J.H.H.)
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Yew S. J. Ho
Department of Molecular Physiology and Biophysics (J.D.C., M.A.B., J.L.W., A.B., J.K., S.H.F.) and Center for Lung Research, Division of Allergy, Pulmonary and Critical Care Medicine (K.P.K.), Vanderbilt University School of Medicine, Nashville, Tennessee; and Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (Y.S.J.H., L.F.S., J.H.H.)
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Layla F. Saidi
Department of Molecular Physiology and Biophysics (J.D.C., M.A.B., J.L.W., A.B., J.K., S.H.F.) and Center for Lung Research, Division of Allergy, Pulmonary and Critical Care Medicine (K.P.K.), Vanderbilt University School of Medicine, Nashville, Tennessee; and Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (Y.S.J.H., L.F.S., J.H.H.)
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James H. Hurley
Department of Molecular Physiology and Biophysics (J.D.C., M.A.B., J.L.W., A.B., J.K., S.H.F.) and Center for Lung Research, Division of Allergy, Pulmonary and Critical Care Medicine (K.P.K.), Vanderbilt University School of Medicine, Nashville, Tennessee; and Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (Y.S.J.H., L.F.S., J.H.H.)
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Jun Kotera
Department of Molecular Physiology and Biophysics (J.D.C., M.A.B., J.L.W., A.B., J.K., S.H.F.) and Center for Lung Research, Division of Allergy, Pulmonary and Critical Care Medicine (K.P.K.), Vanderbilt University School of Medicine, Nashville, Tennessee; and Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (Y.S.J.H., L.F.S., J.H.H.)
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Sharron H. Francis
Department of Molecular Physiology and Biophysics (J.D.C., M.A.B., J.L.W., A.B., J.K., S.H.F.) and Center for Lung Research, Division of Allergy, Pulmonary and Critical Care Medicine (K.P.K.), Vanderbilt University School of Medicine, Nashville, Tennessee; and Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (Y.S.J.H., L.F.S., J.H.H.)
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Abstract

Sildenafil (Viagra) potentiates penile erection by acting as a nonhydrolyzable analog of cGMP and competing with this nucleotide for catalysis by phosphodiesterase-5 (PDE5), but the characteristics of direct binding of radiolabeled sildenafil to PDE5 have not been determined. [3H]Sildenafil binding to PDE5 was retained when filtered through nitrocellulose or glass-fiber membranes. Binding was inhibited by excess sildenafil, 2-(2-methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2-yl)methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T-0156), 3-isobutyl-1-methylxanthine, EDTA, or cGMP, but not by cAMP or 5′-GMP. PDE5 was the only [3H]sildenafil binding protein detected in human lung extract. Using purified recombinant PDE5, [3H]sildenafil exchange dissociation yielded two components with t1/2 values of 1 and 14 min and corresponding calculated KD values of 12 and 0.83 nM, respectively. This implied the existence of two conformers of the PDE5 catalytic site. [3H]Sildenafil binding isotherm of PDE5 indicated KD was 8.3 to 13.3 nM, and low cGMP decreased the KD to 4.8 nM but only slightly increased Bmax to a maximum of 0.61 mol/mol-subunit. Results suggest that these effects occur via cGMP binding to the allosteric cGMP binding sites of PDE5. Results imply that by inhibiting PDE5 and thereby increasing cGMP, sildenafil accentuates its own binding affinity for PDE5, which further elevates cGMP. The data also indicate that after physiological elevation, cGMP may directly stimulate the catalytic site by binding to the allosteric cGMP-binding sites of PDE5, thus causing negative feedback on this pathway.

  • Received November 20, 2002.
  • Accepted March 11, 2003.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 63 (6)
Molecular Pharmacology
Vol. 63, Issue 6
1 Jun 2003
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Research ArticleArticle

[3H]Sildenafil Binding to Phosphodiesterase-5 Is Specific, Kinetically Heterogeneous, and Stimulated by cGMP

Jackie D. Corbin, Mitsi A. Blount, James L. Weeks, Alfreda Beasley, Karl P. Kuhn, Yew S. J. Ho, Layla F. Saidi, James H. Hurley, Jun Kotera and Sharron H. Francis
Molecular Pharmacology June 1, 2003, 63 (6) 1364-1372; DOI: https://doi.org/10.1124/mol.63.6.1364

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Research ArticleArticle

[3H]Sildenafil Binding to Phosphodiesterase-5 Is Specific, Kinetically Heterogeneous, and Stimulated by cGMP

Jackie D. Corbin, Mitsi A. Blount, James L. Weeks, Alfreda Beasley, Karl P. Kuhn, Yew S. J. Ho, Layla F. Saidi, James H. Hurley, Jun Kotera and Sharron H. Francis
Molecular Pharmacology June 1, 2003, 63 (6) 1364-1372; DOI: https://doi.org/10.1124/mol.63.6.1364
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