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 factorlike-stimulated transport, which is activated by cAMP signaling, was unaffected, confirming that only cGMPstimulated 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.

Prolonged presynaptic posttetanic cyclic GMP signaling in Drosophila motoneurons

“…4A; P Ͻ 0.05 for each treatment). Furthermore, transgenic presynaptic expression of cGMP-specific mammalian type 5 PDE (PDE5) (12,13) shortened the epac1-camps response 4-fold and reduced its peak amplitude (Fig. 4B).…”

Behavioral Responses to Hypoxia in Drosophila Larvae Are Mediated by Atypical Soluble Guanylyl Cyclases

“…We reduced cGMP levels by expressing bovine phosphodiesterase 5 (bPDE5, a cGMP-specific PDE) in either or both of these populations of neurons. Previous studies using this construct to express bPDE5 in Malpighian tubules, the osmoregulatory and detoxifying organs of flies (Dow and Davies 2003), resulted in reduced cGMP levels and activity, similar to the effects of sGC inhibitors (Broderick et al 2004).…”

“…The UAS Gyc-88E and UAS CNG RNAi lines were obtained from the Vienna Drosophila RNAi Center (VDRC stocks 21798 for Gyc-88E, and 101745, 29046, 28625, 40964, 100882, 11817, 11816, and 38850 for cng). Generation of the UAS bPDE5 (bovine phosphodiesterase 5) overexpression line and the RNAi lines for the cGMP-dependent protein kinases, dg1 and dg2, have previously been described (Broderick et al 2004;S.-A. Davies, unpublished data).…”

Resolution of the insect ouabain paradox

“…For Na ϩ ,K ϩ ATPase, monoclonal antibody ␣5, developed by D. M. Fambrough (18), was obtained from the Developmental Studies Hybridoma Bank (University of Iowa), and visualized with Texas red anti-mouse. To distinguish Na ϩ ,K ϩ ATPase localization from apical microvilli, tubules were taken from a GFP-fusion gene-trap of the V ATPase vhaSFD subunit (28). For visualization of ouabain transport, tubules were incubated in 2 ϫ 10 Ϫ5 M 9-anthroyl ouabain (Molecular Probes) (29) for 10-15 min, and tubules were examined immediately by confocal microscopy (Zeiss 510 Meta).…”