title: Cellular mechanisms underlying prostaglandin-induced transient cAMP signals near the plasma membrane of HEK-293 cells.
abstract: We have previously used cyclic nucleotide-gated (CNG) channels as sensors to measure cAMP signals in human embryonic kidney (HEK)-293 cells. We found that prostaglandin E(1) (PGE(1)) triggered transient increases in cAMP concentration near the plasma membrane, whereas total cAMP levels rose to a steady plateau over the same time course. In addition, we presented evidence that the decline in the near-membrane cAMP levels was due primarily to a PGE(1)-induced stimulation of phosphodiesterase (PDE) activity, and that the differences between near-membrane and total cAMP levels were largely due to diffusional barriers and differential PDE activity. Here, we examine the mechanisms regulating transient, near-membrane cAMP signals. We observed that 5-min stimulation of HEK-293 cells with prostaglandins triggered a two- to threefold increase in PDE4 activity. Extracellular application of H89 (a PKA inhibitor) inhibited stimulation of PDE4 activity. Similarly, when we used CNG channels to monitor cAMP signals we found that both extracellular and intracellular (via the whole-cell patch pipette) application of H89, or the highly selective PKA inhibitor, PKI, prevented the decline in prostaglandin-induced responses. Following pretreatment with rolipram (a PDE4 inhibitor), H89 had little or no effect on near-membrane or total cAMP levels. Furthermore, disrupting the subcellular localization of PKA with the A-kinase anchoring protein (AKAP) disruptor Ht31 prevented the decline in the transient response. Based on these data we developed a plausible kinetic model that describes prostaglandin-induced cAMP signals. This model has allowed us to quantitatively demonstrate the importance of PKA-mediated stimulation of PDE4 activity in shaping near-membrane cAMP signals.
authors.affiliation: Department of Pharmacology, College of Medicine and Center for Lung Biology, University of South Alabama, Mobile, AL 36688, USA. trich@jaguar1.usouthal.edu
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Rich
authors.forename: Wenkuan
authors.initials: W
authors.lastname: Xin
authors.forename: Celine
authors.initials: C
authors.lastname: Mehats
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Hassell
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Piggott
authors.forename: Xuan
authors.initials: X
authors.lastname: Le
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Karpen
authors.forename: Marco
authors.initials: M
authors.lastname: Conti
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D002021:Buffers; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003198:Computer Simulation; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D006801:Humans; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008954:Models, Biological; D009712:Nucleotides, Cyclic / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011453:Prostaglandins / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020889:Rolipram / Q000494:pharmacology; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002021:Buffers; D007473:Ion Channels; D007546:Isoquinolines; D009712:Nucleotides, Cyclic; D010455:Peptides; D010726:Phosphodiesterase Inhibitors; D011453:Prostaglandins; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; C517139:stearated-Ht31 peptide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D000262:Adenylyl Cyclases; D020889:Rolipram; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide