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, N^G-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).