Receptor-mediated stimulation of lipid signalling pathways in CHO cells elicits the rapid transient induction of the PDE1B isoform of Ca2+/calmodulin-stimulated cAMP phosphodiesterase.
Chinese hamster ovary cells (CHO cells) do not exhibit any Ca2+/calmodulin-stimulated cAMP phosphodiesterase (PDE1) activity. Challenge of CHO cells with agonists for endogenous P2-purinoceptors, lysophosphatidic acid receptors and thrombin receptors caused a similar rapid transient induction of PDE1 activity in each instance. This was also evident on noradrenaline challenge of a cloned CHO cell line transfected so as to overexpress alpha 1B-adrenoceptors. This novel PDE1 activity appeared within about 15 min of exposure to ligands, rose to a maximum value within 30 min to 1 h and then rapidly decreased. In each case, the expression of novel PDE1 activity was blocked by the transcriptional inhibitor actinomycin D. Challenge with insulin of either native CHO cells or a CHO cell line transfected so as to overexpress the human insulin receptor failed to induce PDE1 activity. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1C isoform, did not amplify any fragment from RNA preparations of CHO cells expressing PDE1 activity, although they did so from the human thyroid carcinoma FTC133 cell line. Reverse transcriptase-PCR analyses, using degenerate primers able to detect the PDE1A and PDE1B isoforms, successfully amplified a fragment of the predicted size from RNA preparations of both CHO cells expressing PDE1 activity and human Jurkat T-cells. Sequencing of the PCR products, generated using the PDE1A/B primers, yielded a novel sequence which, by analogy with sequences reported for bovine and murine PDE1B forms, suggests that the PDE1 species induced in CHO cells through protein kinase C activation and that expressed in Jurkat T-cells are PDE1B forms.
Spence
S
S
Division of Biochemistry and Molecular Biology, University of Glasgow, Scotland, U.K.
Rena
G
G
Sullivan
M
M
Erdogan
S
S
Houslay
M D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D003609:Dactinomycin / Q000494:pharmacology; D004790:Enzyme Induction; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry*; D019169:Jurkat Cells; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014162:Transfection
D007527:Isoenzymes; D003609:Dactinomycin; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; C517265:PDE1A protein, human; C517266:PDE1B protein, human; C517272:PDE1C protein, human