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First published online October 21, 2004
Journal of Experimental Biology 207, 4135-4145 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01255

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Conservation of capa peptide-induced nitric oxide signalling in Diptera

Valerie P. Pollock¹, James McGettigan¹, Pablo Cabrero¹, Ian M. Maudlin², 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 Centre for Tropical Veterinary Medicine, Royal School of Veterinary Studies, University of Edinburgh, Edinburgh EH9 1QH, UK

View larger version (42K): [in a new window]   Fig. 2. Expression of nitric oxide synthase (NOS) in insect tubules. Tubules were dissected from the following species: Drosophila melanogaster (A); Aedes aegypti (B); Anopheles stephensi (C); Glossina morsitans (D) and Schistocerca gregaria (E). NOS distribution in intact tubules is shown using anti-uNOS antibody (Broderick et al., 2003; Dow and Davies, 2001; Gibbs and Truman, 1998); cell nuclei were visualised with DAPI (Broderick et al., 2004). Single tubules are shown in each panel, viewed by epifluorescence. Samples were viewed at 10x magnification unless stated otherwise. Tubule diameters can be taken as 35 µm. (Ai) control Drosophila tubule (no antibody); (Aii) pair of tubules showing NOS staining in tubule main segment (m); no staining in initial (i) and lower (l) regions; (Aiii) DAPI staining reveals cell nuclei; (Bi) control A. aegypti tubules (no antibody); (Bii) NOS staining throughout A. aegypti tubule principal cells (excluded stellate cells indicated by yellow arrow); DAPI staining reveals cell nuclei; (Biii) high magnification (50x) showing NOS staining in principal cells; unstained stellate cells indicated by yellow arrows; (Biv) NOS and DAPI-stained tubule, viewed at high magnification (50x); existence of unstained stellate cells confirmed by presence of smaller nuclei compared with principal cells, indicated by arrows; (Ci) control A. stephensi tubules (no antibody); (Cii) NOS staining throughout A. stephensi tubule principal cells (excluded stellate cells indicated by yellow arrow); DAPI staining reveals cell nuclei; (Ciii) high magnification (50x) showing NOS staining in principal cells; unstained stellate cells indicated by yellow arrows; (Civ) NOS and DAPI-stained tubule, viewed at high magnification (50x); existence of unstained stellate cells confirmed by presence of smaller nuclei compared with principal cells, indicated by arrows; (Di) control G. morsitans tubules (no antibody); (Dii) anti-NOS antibody-stained intact G. morsitans tubule at low magnification; DAPI staining reveals cell nuclei; (Diii) anti-NOS antibody-stained tubule at high magnification (20x); (Div) same preparation as Dii, viewed at high magnification (20x); (Ei) control S. gregaria tubules (no antibody) viewed at 20x magnification; (Eii) anti-NOS antibody-stained intact tubule viewed at 20x magnification; close-up view indicates clear staining at the membrane and cytosol; (Eiii) anti-NOS antibody-stained intact tubule viewed at 20x magnification; DAPI staining reveals cell nuclei.

View larger version (35K): [in a new window]   Fig. 3. NADPH diaphorase activity is stimulated by capa peptides. NADPH diaphorase activity was measured in either unstimulated or peptide-stimulated tubules from the insects shown in the absence and presence of the substrate, NADPH (A), as described in Materials and methods. Assessment of NOS-derived NADPH activity was carried out by the inclusion of a nitric oxide synthase (NOS) inhibitor in the assays (B–D). (A) Tubules were stimulated for 10 min by either capa-1 (red), AngCAPA-QGL (blue) or AngCAPA-GPT (green) at a final concentration of 10-7 mol l-1. In order to normalise data for all species, results are expressed as % increase over unstimulated tubules (± S.E.M.; N=6), as described in Materials and methods. (B–D) NADPH diaphorase activity has already been shown to be an accurate estimation of NOS activity in Drosophila melanogaster tubules by use of an inducible transgene for NOS (Broderick et al., 2003). However, in order to investigate a direct correlation of NADPH diaphorase with NOS activity in tubules from the dipteran insects studies here, NADPH diaphorase experiments were performed in the presence of the NOS inhibitor, NG-nitro-L-arginine-methyl ester (L-NAME). This was achieved using tubules from those insect species that showed an increase in NADPH diaphorase activity in A, as follows: (B) tubules from Drosophila melanogaster, Aedes aegypti, Anopheles stephensi and Glossina morsitans were stimulated with capa-1 in the presence of NADPH as above, in the absence (filled bars) or presence (open bars) of L-NAME, and NADPH diaphorase activity was measured. Results are expressed as % increase over control (unstimulated) tubules (± S.E.M.; N=3–8), where control values are 100%. (C) Tubules from the four dipteran species, as above, were stimulated with AngCAPA-QGL in the presence of NADPH as above, in the absence (filled bars) or presence (open bars) of L-NAME, and NADPH diaphorase activity was measured. Results are expressed as % increase over control (unstimulated) tubules (± S.E.M.; N=3–8), where control values are 100%. (D) Tubules from the four dipteran species, as above, were stimulated with AngCAPA-GPT in the presence of NADPH as above, in the absence (filled bars) or presence (open bars) of L-NAME, and NADPH diaphorase activity was measured. Results are expressed as % increase over control (unstimulated) tubules (± S.E.M.; N=3–8), where control values are 100%. *Statistically significant data compared with tubules in the absence of L-NAME, where P<0.05 (Student's t-test, unpaired samples).

View larger version (23K): [in a new window]   Fig. 4. cGMP levels are stimulated by capa peptides in Diptera. Basal and capa-stimulated cGMP levels in tubules from several species were measured by radioimmunoassay (RIA). Tubules were stimulated with capa-1 (red), AngCAPA-QGL (blue) and AngCAPA-GPT (green) peptides (10-7 mol l-1) for 10 min. Data for each sample were calculated as fmol cGMP µg-1 protein (± S.E.M.; N=4–6) in order to normalise the data across species. Protein estimations were conducted by the Bradford assay. In order to aid comparison with other insects, values for G. morsitans tubules have been under-represented on the graph: levels of cGMP in G. morsitans tubules stimulated by A. gambiae capa peptides were 10±0.2 fmol µg-1 protein (AngCAPA-QGL) and 9.8±0.2 fmol µg-1 protein (AngCAPA-GPT) compared with 0.610±0.025 fmol µg-1 protein for control tubules. No increase in cGMP content was observed upon stimulation of S. gregaria tubules with either capa-1, AngCAPA-QGL or AngCAPA-GPT. *Statistically significant data compared with untreated tubules, where P<0.05 (Student's t-test, unpaired samples).

View larger version (15K): [in a new window]   Fig. 1. Expression of capa receptor in dipteran tubules. RT-PCR of tubule cDNA templates with primers designed to the capa receptor gene from D. melanogaster (A, lane 1; product size, 1448 bp), A. aegypti (A, lane 3), A. stephensi (B, lane 1; product size, 250 bp), A. gambiae (B, lane 3; product size, 250 bp). Controls were performed using no cDNA template: D. melanogaster (A, lane 2), A. aegypti (A, lane 4), A. stephensi (B, lane 2), A. gambiae (B, lane 4). L=1 kb ladder. No PCR product was observed with A. aegypti cDNA template. In all cases, products obtained were of the sizes predicted for cDNA templates. Identical PCR products were obtained from both A. gambiae and A. stephensi. Very few A. gambiae were available for study; however, given the documented expression of the putative capa receptor in both species, the more abundant A. stephensi was used for all subsequent experiments.

View larger version (25K): [in a new window]   Fig. 5. Stimulation of fluid transport by capa-1. Tubule fluid secretion was measured in the absence and presence of Drosophila capa-1 at the concentrations shown. Basal rates of secretion were measured for 30 min prior to addition of peptides. Secretion rates were measured for a further 40 min. Data are expressed as percentage stimulation of fluid secretion rate compared with basal rate (± S.E.M.; N=6–8). Statistically significant differences from basal values are denoted by asterisks, where P<0.05 determined by Student's t-test, (unpaired samples). (A) D. melanogaster; (B) A. aegypti; (C) A. stephensi; (D) G. morsitans and (E) S. gregaria.

View larger version (23K): [in a new window]   Fig. 6. Stimulation of fluid transport by AngCAPA-QGL. Tubule fluid secretion was measured in the absence and presence of AngCAPA-QGL at the concentrations shown. Basal rates of secretion were measured for 30 min prior to addition of peptides. Secretion rates were measured for a further 40 min. Data are expressed as percentage stimulation of fluid secretion rate compared with basal rate (± S.E.M.; N=6–8). Statistically significant differences from basal values are denoted by asterisks, where P<0.05 determined by Student's t-test, (unpaired samples). (A) D. melanogaster; (B) A. aegypti; (C) A. stephensi; (D) G. morsitans and (E) S. gregaria.

View larger version (24K): [in a new window]   Fig. 7. Stimulation of fluid transport by AngCAPA-GPT. Tubule fluid secretion was measured in the absence and presence of AngCAPA-GPT at the concentrations shown. Basal rates of secretion were measured for 30 min prior to addition of peptides. Secretion rates were measured for a further 40 min. Data are expressed as percentage stimulation of fluid secretion rate compared with basal rate (± S.E.M.; N=6–8). Statistically significant differences from basal values are denoted by asterisks, where P<0.05 determined by Student's t-test, (unpaired samples). (A) D. melanogaster; (B) A. aegypti; (C) A. stephensi; (D) G. morsitans and (E) S. gregaria.

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