title: Estimating the magnitude of near-membrane PDE4 activity in living cells.
abstract: Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments.
authors.affiliation: Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, South Carolina;
authors.forename: Wenkuan
authors.initials: W
authors.lastname: Xin
authors.affiliation: High Performance Computing, Louisiana State University, Baton Rouge, Louisiana;
authors.forename: Wei P
authors.initials: WP
authors.lastname: Feinstein
authors.affiliation: Department of Pharmacology, University of South Alabama, Mobile, Alabama; and Center for Lung Biology, University of South Alabama, Mobile, Alabama.
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Britain
authors.affiliation: Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas;
authors.forename: Cristhiaan D
authors.initials: CD
authors.lastname: Ochoa
authors.affiliation: Mitchell Cancer Institute, Mobile, Alabama;
authors.forename: Bing
authors.initials: B
authors.lastname: Zhu
authors.affiliation: Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, Alabama; Center for Lung Biology, University of South Alabama, Mobile, Alabama.
authors.forename: Wito
authors.initials: W
authors.lastname: Richter
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of South Alabama, Mobile, Alabama; Department of Pharmacology, University of South Alabama, Mobile, Alabama; and Center for Lung Biology, University of South Alabama, Mobile, Alabama.
authors.forename: Silas J
authors.initials: SJ
authors.lastname: Leavesley
authors.affiliation: Department of Pharmacology, University of South Alabama, Mobile, Alabama; and Center for Lung Biology, University of South Alabama, Mobile, Alabama trich@southalabama.edu.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Rich
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000378:metabolism*; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D015640:Ion Channel Gating / Q000502:physiology; D020889:Rolipram / Q000494:pharmacology; D014970:Xanthines / Q000494:pharmacology
chemical_list: C119531:8-methoxymethyl-3-isobutyl-1-methylxanthine; D054815:Cyclic Nucleotide-Gated Cation Channels; D014970:Xanthines; D000242:Cyclic AMP; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D020889:Rolipram
keywords: CNG channel; G protein-coupled receptor; cAMP compartmentalization; phosphodiesterase; protein kinase A