Ectopic expression of bovine type 5 phosphodiesterase confers a renal phenotype in Drosophila

Kate E Broderick, Laura Kean, Julian A T Dow, Nigel J Pyne, Shireen A Davies

Research output: Contribution to journalArticle

33 Citations (Scopus)

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.
LanguageEnglish
Pages8159-8168
Number of pages10
JournalJournal of Biological Chemistry
Volume279
Issue number9
Early online date8 Dec 2003
DOIs
Publication statusPublished - 2004

Fingerprint

Type 5 Cyclic Nucleotide Phosphodiesterases
Phosphoric Diester Hydrolases
Drosophila
Phenotype
Kidney
Level control
Fluids
Malpighian Tubules
Phosphodiesterase 5 Inhibitors
Corticotropin-Releasing Hormone
Cell membranes
Transgenes
Vertebrates
Ectopic Gene Expression
Western Blotting
Cell Membrane
Polymerase Chain Reaction
Peptides
Sildenafil Citrate

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

Cite this

Broderick, Kate E ; Kean, Laura ; Dow, Julian A T ; Pyne, Nigel J ; Davies, Shireen A. / Ectopic expression of bovine type 5 phosphodiesterase confers a renal phenotype in Drosophila. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 9. pp. 8159-8168.
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Ectopic expression of bovine type 5 phosphodiesterase confers a renal phenotype in Drosophila. / Broderick, Kate E; Kean, Laura; Dow, Julian A T; Pyne, Nigel J; Davies, Shireen A.

In: Journal of Biological Chemistry, Vol. 279, No. 9, 2004, p. 8159-8168.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Ectopic expression of bovine type 5 phosphodiesterase confers a renal phenotype in Drosophila

AU - Broderick, Kate E

AU - Kean, Laura

AU - Dow, Julian A T

AU - Pyne, Nigel J

AU - Davies, Shireen A

PY - 2004

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N2 - 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.

AB - 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.

KW - 3',5'-cyclic-GMP phosphodiesterases

KW - animals

KW - animals, genetically modified

KW - biological transport

KW - blotting, western

KW - cattle

KW - cell membrane

KW - cyclic GMP

KW - cyclic nucleotide phosphodiesterases, type 5

KW - DNA-binding proteins

KW - drosophila

KW - enzyme inhibitors

KW - gene expression

KW - gene targeting

KW - immunohistochemistry

KW - kidney tubules

KW - malpighian tubules

KW - mutagenesis

KW - phenotype

KW - phosphodiesterase inhibitors

KW - phosphoric diester hydrolases

KW - piperazines

KW - purines

KW - recombinant fusion proteins

KW - reverse transcriptase polymerase chain reaction

KW - saccharomyces cerevisiae proteins

KW - sulfones

KW - transcription factors

KW - transfection

U2 - 10.1074/jbc.M304679200

DO - 10.1074/jbc.M304679200

M3 - Article

VL - 279

SP - 8159

EP - 8168

JO - Journal of Biological Chemistry

T2 - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 9

ER -