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First published online July 2, 2004
Journal of Experimental Biology 207, 2769-2776 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01086

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The dg2 (for) gene confers a renal phenotype in Drosophila by modulation of cGMP-specific phosphodiesterase

Matthew R. MacPherson^1,*, Kate E. Broderick^1,*, Shirley Graham¹, Jonathan P. Day¹, Miles D. Houslay², Julian A. T. Dow¹ and Shireen A. Davies^1,

¹ Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, UK
² Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G11 6NU, UK

View larger version (28K): [in a new window]   Fig. 1. Adult forR and fors display distinct locomotor activities. Adult flies were put through activity monitor trials as described. The results show activity over the course of the first day (A) after the initial rest period, of forR (Ai) and fors (Aii) flies, and on day 3 (Bi,ii). Results of analysis of the area under the major peaks (data points 10-25 on day 1; 110-125 on day 3) are shown in (Aiii, Biii). Values (y axis) are arbitary units ± S.E.M. (N=9-10). *P<0.05.

View larger version (13K): [in a new window]   Fig. 2. The fors allele results in a transport phenotype in tubules. Fluid secretion assays were performed on intact tubules from 7-day-old adult forR and fors flies. Basal rates of secretion were measured and capa-1 (10-7 mol l-1) (Kean et al., 2002) added at 30 min (arrow). Secretion rates were monitored for a further 30 min. Fluid secretion rate (nl min-1) are means ± S.E.M. (N=8) for forR tubules (broken line) and fors tubules (solid line). *P<0.05, using t-tests on experimental versus control at each time point separately.

View larger version (12K): [in a new window]   Fig. 3. Cyclic-GMP dependent kinase (cGK) activity in fors tissue. cGK activity was assayed in head, body and tubule preparations from forR and fors 7-day-old adults, as described, in the absence and presence of cGMP. Data was corrected for specific cGMP-dependent activity, and fors data expressed as a % of forR. Values are means ± S.E.M., N=8-12. Mean cGMP-dependent activities (pmol ATP min-1 mg-1 protein) for forR flies were: 15.4±1.3 (heads), 1.1±0.1 (bodies), 12.9±1.5 (tubules); and fors flies: 12.84±1.1 (heads), 1.2±0.1 (bodies), 13.57±2.6 (tubules). In all experiments, cGK activity (pmol ATP min-1 mg-1 protein) was consistently lower in bodies than in either head or tubules.

View larger version (11K): [in a new window]   Fig. 4. fors tubules display increased basal cGMP-phosphodiesterase (cG-PDE) activity. cG-PDE activity was assayed in preparations from head (open bars) and tubule (grey bars) from forR and fors animals as described. To aid comparison between head and tubule samples, cG-PDE activity in forR is taken as 100%, and fors activity normalised against this, for each tissue (means ± S.E.M., N=4-6), *P<0.05. forR cG-PDE activities: head, 211±23 pmol min-1 mg-1; tubule, 642±72 pmol min-1 mg-1 protein.

View larger version (11K): [in a new window]   Fig. 5. Resting cGMP content is reduced in fors tubules. cGMP content of forR (unshaded bars) and fors (grey bars) tubules were assayed (20 per sample) as described. Tubule samples were either untreated or treated with capa-1 (10-7 mol l-1) (Kean et al., 2002) for 10 min prior to terminating the reaction. Data are expressed as cGMP content (fmol 20-1 tubules) mean ± S.E.M., N=4. *Statistically significant data between forR and forS (P<0.05).

View larger version (9K): [in a new window]   Fig. 6. cG-PDE activity is significantly inhibited by capa-1 peptide. cGK (A) and cG-PDE (B) activities were assayed in tubule preparations from forR and fors animals as described, under control and capa-1-stimulated conditions. cGK activity was assessed in the presence of cGMP. Tubules were pre-treated with capa-1 (10-7 mol l-1) (Kean et al., 2002) for 10 min, prior to homogenisation and sample preparation. In order to aid comparison, data for both cGK and cG-PDE activity in the presence of capa-1 are expressed as % of untreated activity, mean ± S.E.M. (N=4-6). *Statistically significant data between controls (100%) and capa-1-treated samples (P<0.05).