2769 An important role of guanosine 3′,5′-cyclic monophosphate (cGMP) in epithelial fluid transport has been demonstrated in the insect equivalent of the vertebrate renal system, the Malpighian tubules (Davies, 2000). Malpighian tubules are fluid transporting, osmoregulatory organs that are critical for insect life (Dow and Davies, 2001). Drosophila melanogaster tubules, which constitute an important genetic model for transporting epithelia (Dow and Davies, 2003), display elevated rates of fluid transport when stimulated by either exogenous cGMP, nitric oxide or neuropeptide-generated nitric oxide/cGMP (Davies et al., 1995, 1997; Dow et al., 1994b; Kean et al., 2002). An autocrine role for NO/cGMP has been proposed for tubule principal cells (Broderick et al., 2003), with NO/GMP signalling being compartmentalised to principal cells in the main, fluid-secreting segment of tubules. These cells contain the electrogenic vacuolar H + -ATPase (V-ATPase) pump (Dow, 1999), which energises fluid transport. Furthermore, electrophysiological studies show that cGMP signalling modulates V-ATPase activity (Davies et al., 1995), suggesting that cGMP signalling may regulate ion transport in tubules. Major effectors of cGMP signalling, including cGMP- dependent protein kinases (cGK) (Vaandrager and de Jonge, 1996) have previously been described in tubules. Furthermore, pharmacological and transgenic modulation of cGMP-specific phosphodiesterase (cG-PDE) activity (Broderick et al., 2004, 2003; Dow et al., 1994b) both result in an epithelial phenotype. In Drosophila, cGK is encoded by two genes, dg1 (Foster et al., 1996) and dg2. Both genes are expressed by Malpighian tubules (Dow et al., 1994b). dg2 was isolated and characterised during a search for cAMP-dependent kinase genes (Kalderon and Rubin, 1989) and the putative cGK shown to be transcribed into three major RNA species of different size and several minor RNA species. These main transcripts (T1, T2 and T3) collectively code for at least three (de Belle et al., 1993), and possibly more, different polypeptides. The DG2 protein shares 64% overall homology with the prototypical bovine lung cGK, with 64% and 75% sequence identity to the cGMP-binding and kinase domains, respectively. Studies in Drosophila have revealed in vivo roles for dg2 The Journal of Experimental Biology 207, 2769-2776 Published by The Company of Biologists 2004 doi:10.1242/jeb.01086 Fluid transport in Drosophila melanogaster tubules is regulated by guanosine 3′,5′-cyclic monophosphate (cGMP) signalling. Here we compare the functional effects on tubules of different alleles of the dg2 (foraging or for) gene encoding a cGMP-dependent protein kinase (cGK), and show that the for s allele confers an epithelial phenotype. This manifests itself as hypersensitivity of epithelial fluid transport to the nitridergic neuropeptide, capa-1, which acts through nitric oxide and cGMP. However, there was no significant difference in tubule cGK activity between for s and for R adults. Nonetheless, for s tubules contained higher levels of cGMP-specific phosphodiesterase (cG-PDE) activity compared to for R . This increase in cGMP-PDE activity sufficed to decrease cGMP content in for s tubules compared to for R . Challenge of tubules with capa-1 increases cGMP content in both for s and for R tubules, although the increase from resting cGMP levels is greater in for s tubules. Capa-1 stimulation of tubules reveals a potent inhibition of cG-PDE in both lines, although this is greater in for s ; and is sufficient to explain the hypersensitive transport phenotype observed. Thus, polymorphisms at the dg2 locus do indeed confer a cGMP-dependent transport phenotype, but this can best be ascribed to an indirect modulation of cG-PDE activity, and thence cGMP homeostasis, rather than a direct effect on cGK levels. Key words: Malpighian tubule, cyclic nucleotide, capa-1, epithelial transport, Drosophila melanogaster, dg2 locus. Summary Introduction 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 Graham 1 , Jonathan P. Day 1 , Miles D. Houslay 2 , Julian A. T. Dow 1 and Shireen A. Davies 1,† Institute of Biomedical and Life Sciences, Divisions of 1 Molecular Genetics and 2 Biochemistry and Molecular Biology, University of Glasgow, Glasgow G11 6NU, UK *These authors contributed equally to this work † Author for correspondence (e-mail: s.a.davies@bio.gla.ac.uk) Accepted 12 May 2004