Broderick, Kate E and Kean, Laura and Dow, Julian A T and Pyne, Nigel J and Davies, Shireen A (2004) Ectopic expression of bovine type 5 phosphodiesterase confers a renal phenotype in Drosophila. Journal of Biological Chemistry, 279 (9). pp. 8159-8168. ISSN 1083-351XFull text not available in this repository. (Request a copy from the Strathclyde author)
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.
|Keywords:||3',5'-cyclic-GMP phosphodiesterases, animals, animals, genetically modified, biological transport, blotting, western, cattle, cell membrane, cyclic GMP, cyclic nucleotide phosphodiesterases, type 5, DNA-binding proteins, drosophila, enzyme inhibitors, gene expression, gene targeting, immunohistochemistry, kidney tubules, malpighian tubules, mutagenesis, phenotype, phosphodiesterase inhibitors, phosphoric diester hydrolases, piperazines, purines, recombinant fusion proteins, reverse transcriptase polymerase chain reaction, saccharomyces cerevisiae proteins, sulfones, transcription factors, transfection, Pharmacy and materia medica, Biochemistry, Cell Biology, Molecular Biology|
|Subjects:||Medicine > Pharmacy and materia medica|
|Department:||Faculty of Science > Strathclyde Institute of Pharmacy and Biomedical Sciences|
|Depositing user:||Pure Administrator|
|Date Deposited:||11 Nov 2011 16:48|
|Last modified:||03 Feb 2017 12:28|