title: Role of phosphodiesterases in the shaping of sub-plasma-membrane cAMP oscillations and pulsatile insulin secretion.
abstract: Specificity and versatility in cyclic AMP (cAMP) signalling are governed by the spatial localisation and temporal dynamics of the signal. Phosphodiesterases (PDEs) are important for shaping cAMP signals by hydrolyzing the nucleotide. In pancreatic β-cells, glucose triggers sub-plasma-membrane cAMP oscillations, which are important for insulin secretion, but the mechanisms underlying the oscillations are poorly understood. Here, we investigated the role of different PDEs in the generation of cAMP oscillations by monitoring the concentration of cAMP in the sub-plasma-membrane space ([cAMP](pm)) with ratiometric evanescent wave microscopy in MIN6 cells or mouse pancreatic β-cells expressing a fluorescent translocation biosensor. The general PDE inhibitor IBMX increased [cAMP](pm), and whereas oscillations were frequently observed at 50 µM IBMX, 300 µM-1 mM of the inhibitor caused a stable increase in [cAMP](pm). The [cAMP](pm) was nevertheless markedly suppressed by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine, indicating IBMX-insensitive cAMP degradation. Among IBMX-sensitive PDEs, PDE3 was most important for maintaining a low basal level of [cAMP](pm) in unstimulated cells. After glucose induction of [cAMP](pm) oscillations, inhibitors of PDE1, PDE3 and PDE4 inhibitors the average cAMP level, often without disturbing the [cAMP](pm) rhythmicity. Knockdown of the IBMX-insensitive PDE8B by shRNA in MIN6 cells increased the basal level of [cAMP](pm) and prevented the [cAMP](pm)-lowering effect of 2',5'-dideoxyadenosine after exposure to IBMX. Moreover, PDE8B-knockdown cells showed reduced glucose-induced [cAMP](pm) oscillations and loss of the normal pulsatile pattern of insulin secretion. It is concluded that [cAMP](pm) oscillations in β-cells are caused by periodic variations in cAMP generation, and that several PDEs, including PDE1, PDE3 and the IBMX-insensitive PDE8B, are required for shaping the sub-membrane cAMP signals and pulsatile insulin release.
authors.affiliation: Department of Medical Cell Biology, Uppsala University, Biomedical Centre Box 571, SE-751 23 Uppsala,  Sweden.
authors.forename: Geng
authors.initials: G
authors.lastname: Tian
authors.forename: Jenny
authors.initials: J
authors.lastname: Sågetorp
authors.forename: Yunjian
authors.initials: Y
authors.lastname: Xu
authors.forename: Hongyan
authors.initials: H
authors.lastname: Shuai
authors.forename: Eva
authors.initials: E
authors.lastname: Degerman
authors.forename: Anders
authors.initials: A
authors.lastname: Tengholm
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000502:physiology*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1 / Q000502:physiology*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000502:physiology*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000502:physiology; D005260:Female; D005947:Glucose / Q000502:physiology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000201:enzymology / Q000378:metabolism; D007527:Isoenzymes / Q000502:physiology; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D010507:Periodicity; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D015290:Second Messenger Systems; D059010:Single-Cell Analysis
chemical_list: D007328:Insulin; D007527:Isoenzymes; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C520790:Pde8a protein, mouse; D005947:Glucose