title: Ectopic expression of bovine type 5 phosphodiesterase confers a renal phenotype in Drosophila.
abstract: cGMP signaling regulates epithelial fluid transport by Drosophila Malpighian (renal) tubules. In order to directly evaluate the importance of cGMP-degrading phosphodiesterases (PDEs) in epithelial transport, bovine PDE5 (a bona fide cGMP-PDE), was ectopically expressed in vivo. Transgenic UAS-PDE5 Drosophila were generated, and PDE5 expression was driven in specified tubule cells in vivo by cell-specific GAL4 drivers. Targeted expression was verified by PCR and Western blotting. Immunolocalization of PDE5 in tubule confirmed specificity of expression and demonstrated localization to the apical plasma membrane. GAL4/UAS-PDE5 tubules exhibit increased cG-PDE activity and reduced basal cGMP levels compared with control lines. We show that wild-type and control tubules are sensitive to the PDE5-specific inhibitor sildenafil and that GAL4/UAS-PDE5 tubules display enhanced sensitivity to sildenafil, compared with controls. cGMP content in GAL4/UAS-PDE5 tubules is restored to control levels by treatment with sildenafil. Thus bovine PDE5 retains cGMP-degrading activity and inhibitor sensitivity when expressed in Drosophila. Expression of PDE5 in tubule principal cells results in an epithelial phenotype, reducing rates of basal and cGMP-/Cardioaccelatory peptide(2b)(CAP(2b))-stimulated fluid transport. Furthermore, inhibition of PDE5 activity by sildenafil restores basal and cGMP-stimulated fluid transport rates to control levels. However, corticotrophin releasing factor-like-stimulated transport, which is activated by cAMP signaling, was unaffected, confirming that only cGMP-stimulated signaling events in tubule are compromised by overexpression of PDE5. Successful ectopic expression of a vertebrate cG-PDE in Drosophila has shown that cG-PDE has a critical role in tubule function in vivo and that cG-PDE function is conserved across evolution. The transgene also provides a generic tool for the analysis of cGMP signaling in Drosophila.
authors.affiliation: Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, USA.
authors.forename: Kate E
authors.initials: KE
authors.lastname: Broderick
authors.forename: Laura
authors.initials: L
authors.lastname: Kean
authors.forename: Julian A T
authors.initials: JA
authors.lastname: Dow
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Pyne
authors.forename: Shireen A
authors.initials: SA
authors.lastname: Davies
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases; D000818:Animals; D030801:Animals, Genetically Modified; D001692:Biological Transport; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D006152:Cyclic GMP / Q000032:analysis / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D004268:DNA-Binding Proteins; D004330:Drosophila / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D018390:Gene Targeting; D007150:Immunohistochemistry; D007684:Kidney Tubules / Q000502:physiology; D008317:Malpighian Tubules / Q000201:enzymology*; D016296:Mutagenesis; D010641:Phenotype; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D011687:Purines; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D000068677:Sildenafil Citrate; D013450:Sulfones; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C052130:GAL4 protein, S cerevisiae; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013450:Sulfones; D014157:Transcription Factors; D000068677:Sildenafil Citrate; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D006152:Cyclic GMP