Estimating the magnitude of near-membrane PDE4 activity in living cells.

Autor:	Xin W; Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, South Carolina;, Feinstein WP; High Performance Computing, Louisiana State University, Baton Rouge, Louisiana;, Britain AL; Department of Pharmacology, University of South Alabama, Mobile, Alabama; and Center for Lung Biology, University of South Alabama, Mobile, Alabama., Ochoa CD; Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas;, Zhu B; Mitchell Cancer Institute, Mobile, Alabama;, Richter W; Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, Alabama; Center for Lung Biology, University of South Alabama, Mobile, Alabama., Leavesley SJ; 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., Rich TC; Department of Pharmacology, University of South Alabama, Mobile, Alabama; and Center for Lung Biology, University of South Alabama, Mobile, Alabama trich@southalabama.edu.
Jazyk:	angličtina
Zdroj:	American journal of physiology. Cell physiology [Am J Physiol Cell Physiol] 2015 Sep 15; Vol. 309 (6), pp. C415-24. Date of Electronic Publication: 2015 Jul 22.
DOI:	10.1152/ajpcell.00090.2015
Abstrakt:	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. (Copyright © 2015 the American Physiological Society.)
Databáze:	MEDLINE
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