Cyclic AMP Control Measured in Two Compartments in HEK293 Cells Phosphodiesterase KM Is More Important than Phosphodiesterase Localization 英文参考文献.docVIP

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Cyclic AMP Control Measured in Two Compartments in HEK293 Cells Phosphodiesterase KM Is More Important than Phosphodiesterase Localization 英文参考文献.doc

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Cyclic AMP Control Measured in Two Compartments in HEK293 Cells Phosphodiesterase KM Is More Important than Phosphodiesterase Localization 英文参考文献

CyclicAMPControlMeasuredinTwoCompartmentsin HEK293Cells:PhosphodiesteraseKM IsMoreImportant thanPhosphodiesteraseLocalization KarinaMatthiesen,JacobNielsen* SynapticTransmission2,H.LundbeckA/S,Copenhagen,Denmark Abstract The intracellular second messenger cyclic AMP (cAMP) is degraded by phosphodiesterases (PDE). The knowledge of individualfamiliesandsubtypesofPDEsisconsiderable,buthowthedifferentPDEscollaborateinthecelltocontrolacAMP signal is still not fully understood. In order to investigate compartmentalized cAMP signaling, we have generated a membrane-targeted variant of the cAMP Bioluminiscence Resonance Energy Transfer (BRET) sensor CAMYEL and have comparedintracellularcAMP measurements withittomeasurementswiththecytosolic BRETsensor CAMYELinHEK293 cells. With these sensors we observed a slightly higher cAMP response to adenylyl cyclase activation at the plasma membrane compared to the cytosol, which is in accordance with earlier results from Fluorescence Resonance Energy Transfer (FRET) sensors. We have analyzed PDE activity in fractionated lysates from HEK293 cells using selective PDE inhibitorsandhaveidentifiedPDE3andPDE10Aasthemajormembrane-boundPDEsandPDE4asthemajorcytosolicPDE. Inhibitionofmembrane-boundorcytosolicPDEscanpotentiatethecAMPresponsetoadenylylcyclaseactivation,butwe seenosignificantdifferencebetweenthepotentiationofthecAMPresponseattheplasmamembraneandincytosolwhen membrane-boundandcytosolicPDEsareinhibited.Whendifferentlevelsofstimulationweretested,wefoundthatPDEs3 and 10 are mainly responsible for cAMP degradation at low intracellular cAMP concentrations, whereas PDE4 is more importantforcontrolofcAMPathigherconcentrations. Citation: Matthiesen K,NielsenJ(2011)CyclicAMPControlMeasuredinTwo Compartments inHEK293Cells:PhosphodiesteraseKM IsMoreImportantthan PhosphodiesteraseLocalization.PLoSONE6(9):e24392.doi:10.1371/journal.pone.0024392 Editor:Karl-WilhelmKoch,UniversityofOldenburg,Germany ReceivedApril26,2011;AcceptedAugust8