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Compartmentalized cyclic adenosine 3',5'-monophosphate at the plasma membrane clusters PDE3A and cystic fibrosis transmembrane conductance regulator into microdomains.

Penmatsa H, Zhang W, Yarlagadda S, Li C, Conoley VG, Yue J, Bahouth SW, Buddington RK, Zhang G, Nelson DJ, Sonecha MD, Manganiello V, Wine JJ, Naren AP - Mol. Biol. Cell (2010)

Bottom Line: Actin skeleton disruption reduces PDE3A-CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A-containing macromolecular complex.Consequently, compartmentalized cAMP signaling is lost.Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA.

ABSTRACT
Formation of multiple-protein macromolecular complexes at specialized subcellular microdomains increases the specificity and efficiency of signaling in cells. In this study, we demonstrate that phosphodiesterase type 3A (PDE3A) physically and functionally interacts with cystic fibrosis transmembrane conductance regulator (CFTR) channel. PDE3A inhibition generates compartmentalized cyclic adenosine 3',5'-monophosphate (cAMP), which further clusters PDE3A and CFTR into microdomains at the plasma membrane and potentiates CFTR channel function. Actin skeleton disruption reduces PDE3A-CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A-containing macromolecular complex. Consequently, compartmentalized cAMP signaling is lost. PDE3A inhibition no longer activates CFTR channel function in a compartmentalized manner. The physiological relevance of PDE3A-CFTR interaction was investigated using pig trachea submucosal gland secretion model. Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion.

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PDE3A inhibition generates compartmentalized cAMP at the plasma membrane of Calu-3 cells. Representative pseudocolor images of CFP/FRET emission ratio before (time = 0 min) and after adding 10 μM cilostazol or 10 μM forskolin (time = 10 min). Look up bar shows magnitude of emission ratio. Line graph is a representative graph for CFP/FRET emission ratio change with time upon adding agonist. Bar graph is mean ratio change ± SEM (n = 6 separate experiments, *p < 0.05).
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Figure 3: PDE3A inhibition generates compartmentalized cAMP at the plasma membrane of Calu-3 cells. Representative pseudocolor images of CFP/FRET emission ratio before (time = 0 min) and after adding 10 μM cilostazol or 10 μM forskolin (time = 10 min). Look up bar shows magnitude of emission ratio. Line graph is a representative graph for CFP/FRET emission ratio change with time upon adding agonist. Bar graph is mean ratio change ± SEM (n = 6 separate experiments, *p < 0.05).

Mentions: To characterize the localization of cAMP upon PDE3A inhibition and to investigate the possible mechanism through which CFTR functionally interacts with PDE3A, a FRET-based cAMP sensor, CFP-EPAC-YFP, was transfected into Calu-3 cells or HEK293 cells and then subjected to ratiometric FRET measurements. This highly sensitive, unimolecular fluorescent cAMP indicator allows to monitor cAMP dynamics in intact cells, with very high temporal and spatial resolution (Ponsioen et al., 2004; Li et al., 2007). As can be seen from Figure 3 and Supplemental Figure S3, in Calu-3 cells, cAMP levels (represented by CFP/FRET emission ratio) increase upon PDE3A inhibition by using cilostazol. More importantly, the increase of cAMP levels occurs mainly at the edge area of the cells, suggesting a highly compartmentalized cAMP accumulation at the plasma membrane. Forskolin was used as a control that elicited a globe increase of cAMP (indicated by the uniform increase of the emission ratio in the entire cytoplasm). We also used this cAMP sensor in HEK293 cells to study if we can monitor a similar response upon PDE3A inhibition. As expected, we observed a dose-dependent increase in compartmentalized cAMP (Supplemental Figure S2). Interestingly, addition of rolipram (10 μM) induces a maximal increase in cAMP levels that is similar to the effect seen with forskolin stimulation (20 μM; Supplemental Figure S2). The data suggests that PDE3A is probably involved in compartmentalized cAMP signaling in these cells.


Compartmentalized cyclic adenosine 3',5'-monophosphate at the plasma membrane clusters PDE3A and cystic fibrosis transmembrane conductance regulator into microdomains.

Penmatsa H, Zhang W, Yarlagadda S, Li C, Conoley VG, Yue J, Bahouth SW, Buddington RK, Zhang G, Nelson DJ, Sonecha MD, Manganiello V, Wine JJ, Naren AP - Mol. Biol. Cell (2010)

PDE3A inhibition generates compartmentalized cAMP at the plasma membrane of Calu-3 cells. Representative pseudocolor images of CFP/FRET emission ratio before (time = 0 min) and after adding 10 μM cilostazol or 10 μM forskolin (time = 10 min). Look up bar shows magnitude of emission ratio. Line graph is a representative graph for CFP/FRET emission ratio change with time upon adding agonist. Bar graph is mean ratio change ± SEM (n = 6 separate experiments, *p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2836961&req=5

Figure 3: PDE3A inhibition generates compartmentalized cAMP at the plasma membrane of Calu-3 cells. Representative pseudocolor images of CFP/FRET emission ratio before (time = 0 min) and after adding 10 μM cilostazol or 10 μM forskolin (time = 10 min). Look up bar shows magnitude of emission ratio. Line graph is a representative graph for CFP/FRET emission ratio change with time upon adding agonist. Bar graph is mean ratio change ± SEM (n = 6 separate experiments, *p < 0.05).
Mentions: To characterize the localization of cAMP upon PDE3A inhibition and to investigate the possible mechanism through which CFTR functionally interacts with PDE3A, a FRET-based cAMP sensor, CFP-EPAC-YFP, was transfected into Calu-3 cells or HEK293 cells and then subjected to ratiometric FRET measurements. This highly sensitive, unimolecular fluorescent cAMP indicator allows to monitor cAMP dynamics in intact cells, with very high temporal and spatial resolution (Ponsioen et al., 2004; Li et al., 2007). As can be seen from Figure 3 and Supplemental Figure S3, in Calu-3 cells, cAMP levels (represented by CFP/FRET emission ratio) increase upon PDE3A inhibition by using cilostazol. More importantly, the increase of cAMP levels occurs mainly at the edge area of the cells, suggesting a highly compartmentalized cAMP accumulation at the plasma membrane. Forskolin was used as a control that elicited a globe increase of cAMP (indicated by the uniform increase of the emission ratio in the entire cytoplasm). We also used this cAMP sensor in HEK293 cells to study if we can monitor a similar response upon PDE3A inhibition. As expected, we observed a dose-dependent increase in compartmentalized cAMP (Supplemental Figure S2). Interestingly, addition of rolipram (10 μM) induces a maximal increase in cAMP levels that is similar to the effect seen with forskolin stimulation (20 μM; Supplemental Figure S2). The data suggests that PDE3A is probably involved in compartmentalized cAMP signaling in these cells.

Bottom Line: Actin skeleton disruption reduces PDE3A-CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A-containing macromolecular complex.Consequently, compartmentalized cAMP signaling is lost.Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA.

ABSTRACT
Formation of multiple-protein macromolecular complexes at specialized subcellular microdomains increases the specificity and efficiency of signaling in cells. In this study, we demonstrate that phosphodiesterase type 3A (PDE3A) physically and functionally interacts with cystic fibrosis transmembrane conductance regulator (CFTR) channel. PDE3A inhibition generates compartmentalized cyclic adenosine 3',5'-monophosphate (cAMP), which further clusters PDE3A and CFTR into microdomains at the plasma membrane and potentiates CFTR channel function. Actin skeleton disruption reduces PDE3A-CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A-containing macromolecular complex. Consequently, compartmentalized cAMP signaling is lost. PDE3A inhibition no longer activates CFTR channel function in a compartmentalized manner. The physiological relevance of PDE3A-CFTR interaction was investigated using pig trachea submucosal gland secretion model. Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion.

Show MeSH
Related in: MedlinePlus