title: Kinetic properties of Ca2+/calmodulin-dependent phosphodiesterase isoforms dictate intracellular cAMP dynamics in response to elevation of cytosolic Ca2+.
abstract: Multiply regulated adenylyl cyclases (AC) and phosphodiesterases (PDE) can yield complex intracellular cAMP signals. Ca2+-sensitive ACs have received far greater attention than the Ca2+/calmodulin-dependent PDE (PDE1) family in governing intracellular cAMP dynamics in response to changes in the cytosolic Ca2+ concentration ([Ca2+]i). Here, we have stably expressed two isoforms of PDE1, PDE1A2 and PDE1C4, in HEK-293 cells to determine whether they exert different impacts on cellular cAMP. Fractionation and imaging showed that both PDEs occurred mainly in the cytosol. However, PDE1A2 and PDE1C4 differed considerably in their ability to hydrolyze cAMP and in their susceptibility to inhibition by the non-selective PDE inhibitor, IBMX and the PDE1-selective inhibitor, MMX. PDE1A2 had an approximately 30-fold greater Km for cAMP than PDE1C4 and yet was more susceptible to inhibition by IBMX and MMX than was PDE1C4. These differences were mirrored in intact cells when thapsigargin-induced capacitative Ca2+ entry (CCE) activated the PDEs. Mirroring their kinetic properties, PDE1C4 was active at near basal cAMP levels, whereas PDE1A2 required agonist-triggered levels of cAMP, produced in response to stimulation of ACs. The effectiveness of IBMX and MMX to inhibit PDE1A2 and PDE1C4 in functional studies was inversely related to their respective affinities for cAMP. To assess the impact of the two isoforms on cAMP dynamics, real-time cAMP measurements were performed in single cells expressing the two PDE isoforms and a fluorescent Epac-1 cAMP biosensor, in response to CCE. These measurements showed that prostaglandin E1-mediated cAMP production was markedly attenuated in PDE1C4-expressing cells upon induction of CCE and cAMP hydrolysis occurred at a faster rate than in cells expressing PDE1A2 under similar conditions. These results prove that the kinetic properties of PDE isoforms play a major role in determining intracellular cAMP signals in response to physiological elevation of [Ca2+]i and thereby provide a rationale for the utility of diverse PDE1 species.
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK.
authors.forename: Tasmina A
authors.initials: TA
authors.lastname: Goraya
authors.forename: Nanako
authors.initials: N
authors.lastname: Masada
authors.forename: Antonio
authors.initials: A
authors.lastname: Ciruela
authors.forename: Debbie
authors.initials: D
authors.lastname: Willoughby
authors.forename: Michael A
authors.initials: MA
authors.lastname: Clynes
authors.forename: Dermot M F
authors.initials: DM
authors.lastname: Cooper
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002457:Cell Extracts; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000201:enzymology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D013347:Subcellular Fractions / Q000187:drug effects; D014970:Xanthines / Q000494:pharmacology
chemical_list: C119531:8-methoxymethyl-3-isobutyl-1-methylxanthine; D002457:Cell Extracts; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D014970:Xanthines; D000242:Cyclic AMP; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; C527988:Pde1A protein, mouse; C517273:Pde1C protein, mouse; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine