Interactions between epithelial nitric oxide signaling and phosphodiesterase activity in Drosophila

Kate E. Broderick,1,* Matthew R. MacPherson,1,* Michael Regulski,2 Tim Tully,2 Julian A. T. Dow,1 and Shireen A. Davies1

1Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, United Kingdom; and 2Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724

Submitted 2 April 2003 ; accepted in final form 30 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Signaling by nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) modulates fluid transport in Drosophila melanogaster. Expression of an inducible transgene encoding Drosophila NO synthase (dNOS) increases both NOS activity in Malpighian (renal) tubules and DNOS protein in both type I (principal) and type II (stellate) cells. However, cGMP content is increased only in principal cells. DNOS overexpression results in elevated basal rates of fluid transport in the presence of the phosphodiesterase (PDE) inhibitor, Zaprinast. Direct assay of tubule cGMP-hydrolyzing phosphodiesterase (cG-PDE) activity in wild-type and dNOS transgenic lines shows that cG-PDE activity is Zaprinast sensitive and is elevated upon dNOS induction. Zaprinast treatment increases cGMP content in tubules, particularly at the apical regions of principal cells, suggesting localization of Zaprinast-sensitive cG-PDE to these areas. Potential cross talk between activated NO/cGMP and calcium signaling was assessed in vivo with a targeted aequorin transgene. Activated DNOS signaling alone does not modify either neuropeptide (CAP2b)- or cGMP-induced increases in cytosolic calcium levels. However, in the presence of Zaprinast, both CAP2b-and cGMP-stimulated calcium levels are potentiated upon DNOS overexpression. Use of the calcium channel blocker, verapamil, abolishes the Zaprinast-induced transport phenotype in dNOS-overexpressing tubules. Molecular genetic intervention in the NO/cGMP signaling pathway has uncovered a pivotal role for cell-specific cG-PDE in regulating the poise of the fluid transporting Malpighian tubule via direct effects on intracellular cGMP concentration and localization and via interactions with calcium signaling mechanisms.

Malpighian tubule; cGMP; calcium; aequorin; CNG channel


THE NITRIC OXIDE (NO) signaling pathway has an important role in maintaining diverse physiological functions in both animals (9, 11, 19) and plants (44). NO, produced by a family of nitric oxide synthase (NOS) enzymes (39), activates its intracellular target, soluble guanylate cyclase (sGC) (38, 46), resulting in increased intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP). Intracellular targets for cGMP may include cGMP-dependent protein kinases (cGKs) and ion channels (28). Termination of cGMP signaling and, hence, of downstream cellular effects is achieved via phosphodiesterases (cG-PDEs). NOS isoforms and activities, as well as guanylate cyclases, have been mapped in vertebrate kidney (2, 40, 43, 45). More recently, a role for inducible NOS (iNOS) in sodium and bicarbonate renal transport has been demonstrated in mouse knockouts for iNOS and endothelial NOS (eNOS) (42).

In invertebrates, NO has been shown to modulate directly fluid transport by the Drosophila Malpighian tubule, an organ critically involved with osmoregulation and ion homeostasis. The tubule is a valuable model for studies of signal transduction and ion transport pathways in an organotypic context and provides one of the most informative phenotypes available for the study of cell signaling in Drosophila (15). By using the molecular genetic tools available in Drosophila, these studies can be applied to genetically identified cell types, thus allowing the analysis of specific pathways in nonexcitable, secretory cells in vivo.

Tubules express a calcium/calmodulin-sensitive Drosophila NOS (DNOS), encoded by the single Drosophila NOS gene, dNOS, (35) in only type I (principal) cells (18). The capa family of nitridergic neuropeptides (capa-1 and capa-2) (20) and the related peptide CAP2b (13) increase the activity of DNOS, with concomitant increases in principal cell intracellular cGMP levels. Use of a sGC inhibitor, methylene blue, inhibits both NO donor- and capa peptide-induced cGMP increases and associated fluid transport (16, 20). Thus NO-induced cGMP signaling occurs in tubules and has a direct effect on fluid transport (Fig. 1).



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Fig. 1. Working model for control of fluid secretion by adult Drosophila melanogaster Malpighian tubules. The routes for cation and ion transport are segregated into separate cell types: the larger principal cell (yellow) and the smaller stellate cell (green). The metabolically active principal cell contains the electrogenic V-type proton-motive ATPase, energizing a putative apical alkali metal/cation exchanger, so achieving net flux of mainly potassium. The stellate cell has a much simpler cyto-architecture and is thought to provide the route for the chloride shunt conductance and water flux. For a review, see Ref. 18. AC, adenylate cyclase; PP, protein phosphatase; CRF, corticotrophin-releasing factor; DLK, Drosophila leucokinin.

 

Directed expression of the calcium reporter, aequorin, to specified tubule cell subtypes in vivo demonstrated that capa peptides also increase cytosolic calcium in only type 1 (principal) cells in the tubule main, fluid-secreting segment (20, 36). As such, activation of both calcium and cGMP signaling pathways occurs during tubule fluid transport.

Although much is known about NOS signaling in tubule, our understanding is predicated on the assumption that NOS levels are stable. However, there is ample evidence that NOS activity varies dynamically. In insects, for example, NOS expression in tubule is known to increase drastically upon ingestion of parasites by mosquitoes (14). How might variation in NOS levels impact upon the control of fluid secretion? In this study, we have utilized transgenic flies that overexpress an inducible dNOS transgene (25) to investigate whether NOS directly determines the transport poise of the tubule or whether regulatory networks regulate NOS signaling.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Drosophila stocks. All strains were maintained on standard Drosophila diet over a 12:12-h light-dark photoperiod at 55% humidity. Strains maintained at 25°C were wild-type Oregon R flies (OrR); c42 GAL4 driver line, used to drive expression of transgenes in only principal cells in tubule main segment (36); and vhaSFD gene-trap line, which expresses GFP-tagged vhaSFD under GAL4 control (31). The dN1-2 line, containing a heat shock-inducible full-length dNOS transgene (second chromosome insertion) (25), was maintained at 18°C.

Animals were subjected to a heat shock regime (15 min at 37°C, 24 h recovery, repeated twice over a total period of 3 days) before experimentation. Heat shock-induced gene expression in Drosophila tissues shows tissue-specific variability, with maximum gene expression in tubules occurring between 4 and 21 h after heat shock (23). Therefore, the heat shock protocol adopted (standard protocol for Drosophila) ensured that sufficient protein was deposited in tubules upon conditional expression of the gene (see RESULTS), without detriment to the animals. Heat-shocked lines (designated hs OrR, hs dN1-2) were allowed to recover at 23°C for 2-3 h before dissections to allow for maximum protein expression. In all experiments, unless otherwise stated, "control" or "control dN1-2" refers to dN1-2 flies reared at 18°C without heat shock.

For calcium experiments, dN1-2 lines were placed in an aequorin background: dN1-2 flies were crossed to a homozygous heat shock (hs) GAL4 aequorin line, denoted aeq; hsGAL4 (29, 36); appropriate progeny were selected and recrossed for several generations until a stable line homozygous for dN1-2 and hsGAL4 was obtained, denoted as aeq; dN1-2;hsGAL4 (APPENDIX). These lines were subjected to heat shock regime (above) before use in calcium experiments; such flies are denoted as aeq;hsdN1-2;hsGAL4.

All flies were used 1 wk postemergence. Flies were cooled on ice and then decapitated before dissection to isolate whole tubules.

Materials. Coelenterazine was purchased from Molecular Probes (Leiden, The Netherlands) and dissolved in ethanol before use. Schneider's medium was obtained from GIBCO Life Technologies (Invitrogen, Paisley, UK). The neuropeptide CAP2b (pyro-ELYAFPRV-amide) (12) was synthesized by Research Genetics (Invitrogen). Protease cocktail inhibitor, verapamil, and Zaprinast were obtained from Calbiochem (Merck Biosciences, Nottingham, UK). All other chemicals were obtained from Sigma (Poole, UK).

Western blot analysis. Western blots using rabbit polyclonal anti-universal NOS antibody (Affinity BioReagents, Cambridge Bioscience, Cambridge, UK) (1:1,000 dilution) were performed according to standard protocols using the Bio-Rad Mini-Protean blotting system. Immunolabeling was visualized using Amersham ECL. Protein samples were prepared from tubules from each line (described under Drosophila stocks), homogenized in ice-cold Tris-lysis buffer (2% wt/vol SDS/70 mM TRIS, pH 6.8, containing protease inhibitor cocktail), and centrifuged at 12,000 g for 5 min to remove debris. Supernatants were assayed for protein concentration (Bradford), and 10-30 µg of protein were run on 4-15% BioRad precast gels. Equal loading of protein in each lane was assessed by results from the Bradford assay before loading and was also assessed by Ponceau S staining after blotting.

Assay for NADPH diaphorase activity in tubule extracts. NADPH diaphorase activity in tubules was measured by a colorimetric assay based on Chiang et al. (7). Intact tubules were dissected from adult flies (lines as described in Fig. 2 legend). Fourteen tubules were used per sample and were placed in 94 µl of 50 mM Tris·HCl, pH 7.4, 1% Triton X, 5 µl of 10 mM XTT [sodium 3'-[(1-phenyl-amino-carbonyl)3,4-tetrazolium] bis(4-methoxy-6-nitro)benzenesulfonic acid], and sodium salt. Samples were incubated at 25°C for 20 min. One microliter of 100 mM NADPH or 1 µl of PBS (for controls without substrate, used as blanks during spectrophotometer readings) were added, and samples were incubated at 25°C for 7 min. Samples were homogenized and made up to 2 ml with distilled water, and colorimetric analysis was performed for all samples by spectrophotometry at 450 nm.



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Fig. 2. The dN1-2 transgene confers increased Drosophila nitric oxide synthase (DNOS) activity in tubules. A: Western blotting of adult Malpighian tubule protein using anti-uNOS antibody. A major band of ~150 kDa represents DNOS. Lanes A and B, control and heat-shocked Oregon R (OrR) samples; lanes C and D, control and heat-shocked dN1-2 samples. Density of bands were quantified by using the gel analysis macro of the ImageJ package (http://rsb.info.nih.gov/ij/docs/menus/analyze.html), after background correction. Values (arbitrary units) were as follows: lane A, 6806; lane B, 4927; lane C, 5708; lane D, 10770. B: assay of NOS activity via nitrite generation in Oregon R (control and heat-shocked) and dN1-2 (control and heat-shocked) tubules. Data are expressed as femtomoles of nitrite produced per tubule ± SE (n = 8). Open bars, control lines; shaded bars, heat-shocked lines. *Statistically significant difference. C: assay of NOS activity via NADPH diaphorase activity in Oregon R (control and heat-shocked) and dN1-2 (control and heat-shocked) tubules. Data are expressed as absorbance at 450 nm per sample of 14 tubules ± SE (n = 8). Open bars, control lines; shaded bars, heat-shocked lines. *Statistically significant difference.

 

Griess assay for nitrite detection. NO generation can be detected by assay of nitrite (a stable metabolite of NO) levels using a modified Griess reaction. Generation of nitrite in tubules was assayed as previously described (20). Briefly, for each sample, 50 intact tubules from adult flies (lines as described in Fig. 2 legend) were dissected into 300 µl of Schneider's medium. Samples were chilled on ice and homogenized. Modified Griess reagent (100 µl; Sigma) and 2.6 ml of water were added to each homogenized sample, and the reactions were incubated at room temperature in the dark for 20 min. Standard curves were generated using sodium nitrite (Sigma) standards in Schneider's medium between 0 and 20 µM.

Absorbance was measured in standards and samples at 540 nm. Concentrations of nitrite in samples were determined from the standard curve, which was linear over the entire concentration range tested.

Immunocytochemistry. Experiments were performed using standard protocols as previously described (30) on fixed, intact tubules from control, hs dN1-2, and vhaSFD tubules. Antibodies used were as follows: rabbit polyclonal anti-universal NOS antibody (Affinity BioReagents), 1:100 dilution; rabbit polyclonal anti-cGMP antibody (Chemicon, Chandlers Ford, UK), 1:3,000 dilution; mouse monoclonal anti-Na+/K+-ATPase antibody (antibody a5, Developmental Studies Hybridoma Bank, University of Iowa), 1:500 dilution; Texas red-labeled goat anti-rabbit antibody, 1:1,000 dilution; and fluorescein-labeled goat anti-rabbit antibody (Diagnostics Scotland), 1:250 dilution. Stained tubules were mounted in VectaShield (Vector Labs, Orton Southgate, UK). To visualize principal cell nuclei, 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) staining was carried out on the vhaSFD tubules after immunocytochemistry by incubating tubules with 1 µg/ml DAPI for 2.5 min. Staining of whole mount tubules was detected by immunofluorescence using a Zeiss Axiocam at x20 magnification.

Tubule cyclic GMP assays. Cyclic GMP levels were measured in pooled samples of 20 tubules dissected from either dN1-2 or hs dN1-2 flies by radioimmunoassay (Amersham Biotrak Amerlex M) as previously described (12). Where required, tubules were preincubated with the PDE inhibitor, Zaprinast, at 10-5 M for 10 min. Incubations were terminated with ice-cold ethanol and homogenized. Samples were dried down and resuspended in 0.05 M sodium acetate buffer (Amersham) and processed for cGMP content according to manufacturer's protocol.

Fluid secretion assays. Malpighian tubules were isolated from control and hs dN1-2 flies into 10-µl drops of Schneider's medium under liquid paraffin, and fluid secretion rates were measured in tubules as detailed elsewhere (16) under various conditions, as described in the text.

Assay for tubule cG-PDE activity. cG-PDE activity in tubules was assayed according to protocols for cAMP-dependent PDE activity in this tissue (6). For each sample, 50 tubules (20-30 µg of protein) from each line were dissected into 50 µl of PBS (pH 7.4) containing protease inhibitor cocktail (Sigma) and homogenized. Fifty microliters of tritiated cGMP working stock (0.185 kBq/ml in 20 µM cGMP, 10 mM Tris, 5 mM MgCl2, pH 7.4) were added to each sample on ice. Blank samples were prepared using 50 µl of PBS and 50 µl of working stock; positive control was made as blanks except that PBS was replaced by bovine PDE5-transformed cell lysates (10).

The samples were incubated at 30°C for 10 min, and reactions were terminated by boiling for 2 min. Samples were chilled on ice and incubated for 10 min with 25 µl of 1 mg/ml Crotalus atrox 5'-nucleotidase (Sigma) to allow conversion of guanosine monophosphate to guanosine. Four hundred microliters of resuspended (1:2 vol/vol in water) DOWEX 1-Cl resin (Sigma) were added to each sample, and samples were vortexed every 5 min for 15 min. The samples were centrifuged at 12 000 g for 2 min and 150 µl of the supernatant was removed and added to 2 ml of Optiflow scintillant. Final activity was expressed per milligram of protein by dividing by the amount of protein in the sample assayed. Protein concentrations were assayed according to standard protocols (Lowry).

Measurements of intracellular calcium concentration [Ca2+]i. For each assay, 20-40 tubules from 4- to 14-day-old adults were dissected in Schneider's medium 2 h after final heat shock. Tubules were pooled in 160 µl of the same buffer containing the apoaequorin bioluminescent substrate, coelenterazine (2.5 µM final concentration); reconstitution of aequorin occurred upon incubation in the dark for 3-4 h (36). Bioluminescence recordings were made using a luminometer (LB9507, Berthold, Pforzheim, Germany); recordings were made every 0.1 s for each tube. Each sample was used for a single data point: after recording [Ca2+]i levels, tissues were disrupted in 350 µl of lysis solution [1% (vol/vol) Triton X-100/100 mM CaCl2], causing discharge of the remaining aequorin and allowing estimation of the total amount of aequorin in the sample. Calcium concentrations were calculated as previously described (36). Mock injections with Schneider's medium were applied to all samples before treatment with either neuropeptide (30, 36) or cGMP (30) as previously described.

Statistics. Data are presented as means ± SE. Where appropriate, the significance of differences between data points was analyzed using Student's t-test for unpaired samples, taking P < 0.05 as the critical level.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Activation of NOS signaling in tubules using a dNOS transgene. Although dNOS overexpression via the dN1-2 transgene has been shown in the eye (25), it is important to confirm that driving the NOS transgene overexpresses DNOS as intended. Three different assays conducted on tubules from wild-type, control, and hs dN1-2 lines (Western blotting, measurement of nitrite generation, and an NADPH diaphorase assay) show that overexpression of DNOS, with corresponding increase in DNOS activity, occur upon transgene induction.

Western blotting showed that DNOS protein levels increase in dN1-2 tubules upon heat shock (Fig. 2A). DNOS expression levels did not change in Oregon R upon heat shock, and expression levels were similar to those in control dN1-2 tubules. However, hs dN1-2 tubules (Fig. 2A, lane D) showed higher levels of DNOS expression compared with controls.

Status of tubule DNOS activity in wild-type and dN1-2 flies was assessed using two assays for NOS activity. Oregon R tubules did not exhibit increased nitrite generation upon heat shock (Fig. 2B), whereas hs dN1-2 tubules showed elevated nitrite generation compared with controls. Similarly, using an indicator of NOS activity, NADPH diaphorase activity, only hs dN1-2 tubules showed increased activity compared with either wild-type or non-heat-shocked controls (Fig. 2C). Thus three independent lines of experimentation confirm that induction of the dNOS transgene did result in significant activation (by around 100%) of NO signaling in tubules, as would be expected from previous work with these lines (25).

In tubules, DNOS is normally localized to principal cells in the main, fluid-secreting segment (Ref. 18 and Fig. 3A). Heat shock-driven dN1-2, which would be expected to overexpress DNOS ubiquitously, indeed resulted in increased DNOS protein in both principal and stellate cells, as assessed by immunostaining (Fig. 3, A and B). However, in spite of ubiquitous expression of DNOS in tubule, cGMP only increased in principal cells (Fig. 3, C and D), suggesting that only this cell type is competent to receive NO signals, via sGC, as would be required for NO signal transduction. Thus it is likely that NO is produced and utilized for cGMP signaling only by principal cells in vivo.



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Fig. 3. NO/cGMP signaling is confined to principal cells in tubules. A and B: immunolocalization of DNOS in dN1-2 (A) and hs dN1-2 (B) tubules, using anti-uNOS antibody and Texas Red secondary antibody, visualized by epifluorescence microscopy. Control showing tubules without primary antibody but with Texas red secondary antibody is shown in E. C and D: immunolocalization of cGMP in dN1-2 (C) and hs dN1-2 (D) tubules, using anti-cGMP antibody and fluorescein secondary antibody, visualized by epifluorescence microscopy. Control showing tubules without primary antibody but with fluorescein secondary antibody is shown in F. Where visible, stellate cells are arrowed. -hs indicates dN1-2 lines, whereas +hs indicates hs dN1-2. For comparison, all samples were processed in parallel and photographed with a Zeiss Axiocam under identical exposure settings. These tubules are representative of a large set of experiments. In all micrographs, the scale is given by the tubule diameter, which can be taken to be 35 µm.

 

cG-PDE inhibitor unmasks an epithelial phenotype conferred by dNOS overexpression. Activation of NO signaling in tubules either by NO donors (16) or capa peptides (20) is associated with an increase in epithelial fluid transport. However, manipulation of NO signaling via the dNOS transgene over a twofold range (Fig. 2) did not promote any significant change in fluid transport (Fig. 4A). By contrast, in the presence of Zaprinast, a substantial increase in basal rates of fluid transport (1.51 ± 0.32 fold over control levels) was observed in hs dN1-2A tubules compared with Zaprinast-treated control tubules (Fig. 4B). Finally, dNOS overexpression does not compromise the normal tubule secretion response to CAP2b: both normal and heat-shocked tubules produced a similar absolute response to CAP2b (Fig. 4, A and B).



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Fig. 4. The cG-phosphodiesterase (PDE) inhibitor, Zaprinast, unmasks an epithelial phenotype for DNOS overexpression. A and B: Zaprinast elevates basal rates of fluid transport; tubules from control dN1-2 ({blacksquare}) and hs dN1-2 ({bullet}) lines assayed for fluid transport phenotype, in the absence (A) and presence (B) of Zaprinast (10-5 M). Tubules were pretreated with Zaprinast for 10 min before stimulation with CAP2b at 20 min (indicated by arrow). Data are expressed as mean fluid secretion rate (nl/min) ± SE (n = 8). C: Zaprinast treatment (10-5 M) elevates cGMP levels both in control and heat-shocked dN1-2 tubules. cGMP levels are also elevated upon dN1-2 expression. cGMP levels were measured by radioimmunoassay (RIA), and data are expressed as femtomoles of cGMP per 20 tubules ± SE, (n = 4-6). Although cGMP content was measured in homogenized tubule samples, these measurements represent cGMP content of principal cells because stellate cells do not generate cGMP under these conditions. *Significant difference in cGMP content between untreated and Zaprinast-treated samples (P < 0.05). D: immunolocalization of cGMP with anti-cGMP antibody and fluorescein secondary antibody in dN1-2 tubules that have either been untreated (i) or treated (ii) with Zaprinast. Figure 3 (F) shows control tubule with no primary antibody. iii and iv: hs dN1-2 tubules that have been untreated (iii) or treated (iv) with Zaprinast. v provides orientation: the narrow apical domain is marked with GFP-tagged vhaSFD (green), and the deep basolateral infoldings are stained with anti-Na+/K+-ATPase antibody (37), using a Texas red secondary (red). Cell nuclei are stained with DAPI (blue). For clarity, panels have been labeled with combinations of -hs (dN1-2), +hs (hs dN1-2), -Zap (tubules untreated with Zaprinast), and +Zap (tubules treated with Zaprinast). For comparison, all samples were processed in parallel and photographed with a Zeiss Axiocam under identical exposure settings. These tubules are representative of a large set of experiments. In all micrographs, the scale is given by the tubule diameter, which can be taken to be 35 µm.

 

cGMP levels were massively increased upon dNOS transgene overexpression (Fig. 4C). Basal cGMP levels were elevated 10-fold (9.15 ± 1.08) in hs dN1-2 compared with controls. The cGMP-PDE inhibitor, Zaprinast, significantly potentiated this response (13.5 ± 2.5-fold increase over controls; Fig. 4C). CAP2b also elevates cGMP levels in hs dN1-2 tubules (dN1-2 + CAP2b: 150 ± 7.4 fmol/20 tubules; dN1-2 + CAP2b + Zaprinast: 171 ± 1.31 fmol/20 tubules; hs dN1-2 tubules + CAP2b: 181 ± 3 fmol/20 tubules; hs dN1-2 + CAP2b + Zaprinast: 195 ± 12 fmol/20 tubules). This may explain the stimulated fluid secretion observed in Fig. 4, A and B.

Zaprinast also caused a small but significant elevation in cGMP levels in control dN1-2 tubules (Fig. 4C), as is observed in wild-type tubules (5).

Spatial distribution of cytosolic cGMP is altered by Zaprinast. Although NOS overexpression drives cGMP to very high levels, this does not necessarily increase fluid secretion. The action of Zaprinast, in increasing fluid secretion while only providing a minor further increase in cGMP, suggested that the main targets of cGMP signaling in principal cells were shielded from changes in bulk cytosolic cGMP by a Zaprinast-sensitive PDE. Accordingly, we investigated the spatial distribution of cGMP in intact tubules after Zaprinast treatment.

cGMP was localized using immunocytochemistry in either Zaprinast-treated or untreated control and hs dN1-2 tubules (Fig. 4D). For comparison, the apical and basolateral membrane in tubules were marked using GFP-tagged V-ATPase SFD subunit (apical) (31) and anti-Na+/K+-ATPase (basolateral) (37) (Fig. 4Dv). In control dN1-2 tubules (Fig. 4Di), Zaprinast treatment resulted in an overall increase in cGMP content, with accumulation at or near the membranes of principal cells (Fig. 4Dii). Increased cGMP content was also seen at membranes of principal cells in Zaprinast-treated hs dN1-2 tubules (Fig. 4, Div vs. Diii). This was not as marked as in control dN1-2 tubules because of increased cytosolic staining of cGMP upon dN1-2 induction. By inspection, the localization of cGMP at the membrane is similar to that of apical vhaSFD localization observed using tubules from the vhsSFD gene-trap line (Fig. 4, Dv vs. Diii/iv). Furthermore, compared with Na+/K+-ATPase localized to the basolateral membrane (Fig. 4Dv), it is clear that Zaprinast induces an increase in cGMP in the vicinity of the apical membrane.

These data suggest that subcellular localization of cGMP is an important determinant of the transport phenotype in tubules. However, bulk concentration of cGMP is also important, because the marked Zaprinast-induced cGMP increase at the apical membrane in control dN1-2 tubules does not result in a transport phenotype.

cG-PDE activity is increased upon dNOS activation. Given that cGMP levels are increased upon Zaprinast treatment in both control and hs dN1-2 tubules, it is very likely that the poise of the NO-signaling pathway is limited by cG-PDE. Accordingly, cG-PDE activity was measured in tubule homogenates prepared from wild-type (OrR), dN1-2, and hs dN1-2 tubules, which were either untreated or treated with Zaprinast. Results (Fig. 5) show that basal cG-PDE activities were similar in wild-type and control dN1-2 lines and that the cG-PDE activity was significantly inhibited by Zaprinast to similar extents (Fig. 5A). In hs dN1-2, however, cG-PDE was increased (more than sevenfold) upon dNOS induction (Fig. 5B). Zaprinast treatment also substantially reduced cG-PDE activity in hs dN1-2 tubules.



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Fig. 5. Zaprinast inhibits tubule cG-PDE activity; DNOS overexpression increases cGPDE activity. A: measurement of cGMP hydrolysis was carried out in tubule homogenates prepared from Oregon R and control dN1-2 tubules. Tubules were either treated or untreated with Zaprinast (10-5 M) for 10 min. cG-PDE activity is expressed in pmol GMP·min-1·mg protein-1 ± SE (n = 4-6). B: as in A but with heat-shocked dN1-2 tubules only. Note that y-axis units of cG-PDE activity are 10-fold higher than in A. *Statistically significant difference.

 

These data show that overexpression of NOS elevated cG-PDE activity, so limiting the physiological effects of enhanced NO signaling. Moreover, the inhibition of a substantial fraction of tubule cG-PDE activity by Zaprinast resulted in increased cGMP concentration at specific cellular locations, resulting in the Zaprinast-induced transport phenotype observed upon induction of the dNOS transgene.

Cross talk between NO/cGMP and calcium signaling pathways. Previous work showed that the cyclic nucleotide-gated calcium channel gene, cng, is expressed in tubule, suggesting that cGMP acts to raise [Ca2+]i (30). Furthermore, capa neuropeptides (Drosophila capa-1, capa-2, and Manduca sexta CAP2b) elevate both [Ca2+]i and NO/cGMP in tubule principal cells (20). Accordingly, we examined possible cross talk between endogenously activated NO/cGMP signaling and calcium signaling pathways in principal cells of dN1-2 tubules (Figs. 6 and 7). To achieve this, we generated flies homozygous for the dN1-2 transgene in an aequorin background, with both transgenes driven by heat shock: aeq;hsdN1-2;hsGAL4 (APPENDIX). Although heat shock drives expression of the aequorin transgene ubiquitously (36), it is nonetheless possible to monitor [Ca2+]i changes in only principal cells of isolated tubules, because exogenous CAP2b and cGMP act only on this cell type (30, 36). Thus comparison with a principal cell-specific line, for example, c42;aeq (36), should give qualitatively comparable results. This was indeed the case for all experiments shown in Fig. 6 (for CAP2b response, see Ref. 25; for cGMP response, see Ref. 30 and Fig. 7). (See next section.)



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Fig. 6. Activated NO/cGMP signaling does not affect calcium signaling in principal cells. CAP2b-induced and cGMP-induced increase in cytosolic calcium levels ([Ca2+]i) are not affected by DNOS overexpression. A and B: representative traces of 10-7 M CAP2b-stimulated [Ca2+]i are shown in control aeq;hsGAL4 (A) and aeq;hsdN1-2;hsGAL4 (B) tubules. For measurements of [Ca2+]i, 20 tubules per sample were stimulated with 10-7 M CAP2b after a mock injection with Schneider's medium. Data are expressed as [Ca2+]i (nM) against time (s); each data point corresponds to 0.1 s and is typical of 5 experiments; addition of stimulus is indicated by the arrows. A biphasic rise in [Ca2+]i is observed (20). C and D: representative traces of cGMP-stimulated [Ca2+]i are shown (30) in control aeq;hsGAL4 (C) and aeq;hsdN1-2;hsGAL4 (D) tubules. Measurements of intracellular calcium were performed as above, with application of cGMP (10-4 M) denoted by an arrow. Data are representative of 12 experiments.

 


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Fig. 7. Inhibition of cG-PDE activity potentiates calcium signaling. Pooled data of CAP2b- and cGMP-stimulated [Ca2+]i increases in the absence (open bars) or presence (shaded bars) of Zaprinast (10-5 M) are shown at maximum time points (450 s) from control, aeq;hsGAL4 (A and C); and aeq;hsdN1-2; hsGAL4 (B and D). Zaprinast-treated tubules were incubated with the drug for 10 min before application of either CAP2b (10-7 M) (A and B) or cGMP (10-4 M) (C and D). Measurements of intracellular calcium were performed as for those in Fig. 6. Data are expressed as [Ca2+]i (nM) (stimulated-average basal reading) ± SE (n = 5-8). For CAP2b stimulations, only data for the secondary rise are presented because no change in magnitude/kinetics of primary response was detected. Pooled values for CAP2b-stimulated [Ca2+]i primary response in the presence of Zaprinast (nM ± SE; n = 5): 172 ± 30.5 (aeq; hsGAL4); 179.2 ± 11 (aeq;hsdN1-2;hsGAL4). Compare to non-Zaprinast-treated samples, described in RESULTS for Fig. 6.

 

Does endogenous generation of NO impact on capa (CAP2b)-stimulated calcium signaling in tubule? Increased levels of NOS and cGMP in vivo did not affect the CAP2b-induced [Ca2+]i rise in principal cells (Fig. 6, A and B). Typical traces for CAP2b-induced calcium increases in principal cells of wild-type (aeq;hsGAL4, Fig. 6A and aeq;hsdN1-2A;hsGAL4, Fig. 6B) tubules are shown. Pooled values for CAP2b-stimulated [Ca2+]i primary responses (nM ± SE, n = 5) were 197.4 ± 20.5 (aeq;hsGAL4); 175.5 ± 40.4 (aeq;hsdN1-2;hsGAL4); pooled values for secondary response (nM ± SE, n = 5) were 41.6 ± 7.5 (aeq;hsGAL4); 38.4 ± 7.1 (aeq; hsdN1-2;hsGAL4).

Application of exogenous cGMP to intact tubules has been shown to induce a slow rise in principal cell [Ca2+]i (30). However, manipulation of endogenous cGMP upon dN1-2 induction did not significantly alter the rise in [Ca2+]i (Fig. 6, C and D). Typical traces for cGMP-induced calcium increases in principal cells of wild-type (aeq; hsGAL4, Fig. 6C) and aeq;hsdN1-2; hsGAL4, (Fig. 6D) tubules are shown. Pooled values for cGMP-stimulated [Ca2+]i (nM ± SE, n = 12) are 29 ± 4.7 (aeq;hsGAL4); 25.5 ± 5.5 (aeq;hsdN1-2;hsGAL4). dN1-2 induction appeared to alter the kinetics of the cGMP-induced [Ca2+]i response, where the peak increase in [Ca2+]i occurred between 120-130 s, compared with ~230 s in controls (Fig. 6, C and D). However, analysis of pooled data from all samples indicated that these differences were not statistically significant.

Thus activated NO/cGMP signaling alone does not impact on calcium signaling in principal cells.

Zaprinast increases cGMP-mediated calcium signaling. Data in Fig. 6 showed that activated NO signaling has no effect on calcium signaling in tubules. However, because Zaprinast inhibition of cG-PDE uncovers a role for activated NO signaling in fluid transport, we investigated a possible role for cG-PDE in calcium signaling by using Zaprinast.

Figure 7 shows CAP2b- and cGMP-stimulated [Ca2+]i levels in Zaprinast-treated and untreated tubules from control and aeq;hsdN1-2;hsGAL4 lines. Results show the total calcium response to cGMP (Fig. 7, C and D) and of the secondary rise stimulated by CAP2b (Fig. 7, A and B).

CAP2b-induced primary [Ca2+]i responses were not affected by either activated NO/cGMP signaling nor Zaprinast treatment (Fig. 6, and data not shown). However, whereas the secondary rise of the CAP2b-induced biphasic [Ca2+]i response in wild-type tubules was not significantly affected by inhibition of cG-PDE (Fig. 7A), in aeq;hsdN1-2;hsGAL4, the secondary [Ca2+]i signal was significantly potentiated by Zaprinast treatment (Fig. 7B). Thus the secondary calcium rise induced by CAP2b is dependent on cGMP.

Treatment of aeq;hsGAL4 and aeq;hsdN1-2; hsGAL4 tubules with Zaprinast alone showed that potentiation of the cGMP-induced [Ca2+]i signal only occurred when dNOS is overexpressed (Fig. 7, C and D).

In c42;aeq, the CAP2b-induced secondary rise in [Ca2+]i signal was not significantly affected by Zaprinast: 105.4 ± 7.3 vs. 123.3 ± 5.7 nM ± SE (n = 8), whereas the cGMP-induced [Ca2+]i rise was similarly not significantly affected by Zaprinast treatment: 27.4 ± 4.7 vs. 45.83 ± 8.1 nM ± SE (n = 8). These results are similar to those obtained for aeq; hsGAL4, which confirms that the use of this line in conjunction with either CAP2b or cGMP faithfully reports [Ca2+]i signaling processes in principal cells.

These data show that Zaprinast acts to increase cGMP-induced calcium signaling in principal cells via increased concentration of cGMP at the apical membrane. As such, cG-PDE(s) may act to maintain stimulated calcium increases at wild-type levels upon dNOS induction.

The Zaprinast-induced transport phenotype in hs dN1-2 tubules is inhibited by verapamil. We have shown that Zaprinast treatment results in increased basal fluid secretion rates in hs dN1-2 tubules. Given that Zaprinast treatment also results in a distinct increase in cGMP at the apical membrane and increases cGMP-dependent [Ca2+]i, we examined a possible link between cGMP and calcium at this cellular location. Previous work has shown that the cGMP-induced rise in fluid transport and [Ca2+]i signal is verapamil sensitive (30). Furthermore, use of high concentrations of verapamil results in binding to the apical membrane (30). As such, there is the possibility that Zaprinast treatment allows activation of verapamil-sensitive channels, resulting in increased [Ca2+]i and the transport phenotype observed.

Figure 8 shows the elevation in basal rates of fluid transport observed in hs dN1-2 tubules upon Zaprinast treatment. Verapamil treatment of these tubules did not affect basal rates compared with untreated tubules. However, verapamil completely abolished the Zaprinast-induced increase in fluid transport rates. Thus the epithelial phenotype observed in Zaprinast-treated hs dN1-2 tubules may involve cGMP-activated verapamil-sensitive calcium channels.



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Fig. 8. Verapamil abolishes the Zaprinast-induced epithelial phenotype in hs dN1-2 tubules. Basal fluid secretion rates were measured in intact tubules from hs dN1-2 flies over a 40-min period. Tubules were either untreated ({triangleup}) or pretreated with Zaprinast (10-5 M) ({bullet}), verapamil (10-3 M) ({diamondsuit}) or with both Zaprinast and verapamil ({blacksquare}). Data are expressed as mean fluid secretion rate (nl/min) ± SE (n = 8). Zaprinast significantly elevates basal secretion rates. No significant differences in secretion rates were observed between untreated, verapamil-, or Zaprinast and verapamil-treated tubules.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Nitric oxide signaling in principal cells. Previous work demonstrated a direct role for NO/cGMP signaling in tubule function (12, 13, 16). We show here that molecular genetic intervention in the NO/cGMP signaling pathway uncovers an important role for cG-PDE in tubule function, where NO has an autocrine signaling role.

The localization of DNOS and capa-stimulated cGMP in principal cells (18, 20) shows that in tubules, it is these cells that utilize the NO signaling pathway. However, although this is persuasive evidence that NO may be an autocrine signaling molecule in tubules, this does not exclude the possibilities that either NO produced in the principal cell may be transduced in neighboring stellate cells or that receptors for capa peptides are principal cell specific. We show here that induction of the dNOS transgene results in expression of DNOS in both principal and stellate cells, although only principal cells show increased cGMP content (Fig. 3). This suggests that stellate cells cannot respond to an intracellular NO signal and are thus unlikely to respond to a paracrine signal from the principal cell. Application of the NO donor, sodium nitroprusside, to intact tubules, showed that a NO-induced cGMP increase is only observed in principal cells (5). Thus NO is used as an autocrine signaling molecule by principal cells. Application of cGMP to tubules increases the transepithelial potential (12), indicative of activation of vacuolar H+-ATPase (V-ATPase), which is localized to principal cells (17). Thus a major function of NO/cGMP signaling is to regulate cation transport in this cell type. A similar transport regulatory role for NO has been proposed for vertebrate macula densa, where autocrine production of NO may inhibit sodium/potassium/chloride cotransport (43). NO has also been shown to inhibit the sodium/potassium ATPase, thus limiting sodium chloride absorption into the macula densa (27).

Role of cG-PDE in fluid transport. Intriguingly, generation of high concentrations of cGMP in tubules as a result of increased DNOS expression does not cause increased fluid transport unless the cG-PDE inhibitor, Zaprinast, is present. Given that stimulated cGMP in principal cells is very high (Fig. 4C), resulting in an estimated concentration of 10 µM cGMP in tubule main segment, the regulation of intracellular cGMP levels by cG-PDE is of real importance in this cell type. The phenotype observed using the heat shock-inducible dN1-2 transgene has been replicated using the GAL4-UAS system, where NOS transgene expression is specifically targeted to principal cells in the living animal, using the c42 GAL4 driver (Broderick, Lynch, and Davies, unpublished observations). Thus the phenotype described in this paper is not an artifact of the inducible system nor of the heat shock regime used.

We have made the first measurements of cG-PDE activity in any insect tubule and have shown that ~50% of cG-PDE activity is inhibited by Zaprinast in vivo; furthermore, induction of the dNOS transgene potentiates tubule cG-PDE activity (Fig. 5). Measurements of cGMP-dependent protein kinase (cGK) activity in the absence and presence of cGMP in hs dN1-2 tubules have shown that, surprisingly, cGK activity is reduced upon DNOS overexpression: dN1-2: 11.2 ± 0.6; hs dN1-2: 6.7 ± 0.5 (data expressed as picomoles of ATP per minute per milligram of protein ± SE, n = 5). cGK and cG-PDE have been shown to compete for cGMP (10), and it may be that the very active cG-PDE in tubules reduces availability of cGMP to cGK, with consequential loss of cGMP-induced kinase activity.

Thus hydrolysis of cGMP via an active cG-PDE is a major factor in maintaining stability in fluid transport rates in response to NO. The role of cG-PDE in stabilizing tissue responses to NO is supported by recent work in human intact platelets (32), which has shown that NO/cGMP signaling enhances PDE5 activity. cGMP has been shown to directly activate PDE5 activity without the involvement of cGK and provides a mechanism of negative feedback to limit the effects of activated NO/cGMP signaling in platelets.

Other work has suggested that cG-PDE5 is pivotal in maintaining cGMP levels. cGMP levels are regulated in gastric smooth muscle by activation of PDE5 and inhibition of soluble guanylate cyclase (33). Also, in rat small intestine, NO-induced vasopressin release is modulated by cGK and PDE5 (24). Thus PDE5, as opposed to sGC, may be of particular importance in maintaining critical cGMP levels in epithelia and nonexcitable tissue.

Use of inhibitors of PDE isoenzymes has revealed compartmentalization of cAMP signaling in nonexcitable cells in vertebrates, including mesangial cells (8) and oocytes (41). This also implies that PDEs themselves are localized at discrete subcellular locations. We show here that the PDE inhibitor Zaprinast causes accumulation of cGMP at the apical membrane, where the V-ATPase is located (Fig. 4Dv and Refs. 21, 34, 17). Data presented here strongly suggest that Zaprinast-sensitive cG-PDE(s) exist at the apical regions in tubule principal cells, thus providing specificity of cGMP signaling in the vicinity of the V-ATPase and linking cGMP signaling with increased fluid transport.

The Zaprinast-induced transport phenotype is associated with verapamil-sensitive calcium channels. Interactions between cGMP and calcium signaling pathways have been observed in several vertebrate systems, including the visual system (22) and in smooth muscle (1). However, although the role of cGMP/calcium cross talk in renin secretion has been investigated (3), the direct effects of cGMP/calcium-signaling mechanisms in a renal context in vertebrates have not been fully explored.

We have shown that both cGMP and calcium signaling are important in tubule function. In particular, the capa neuropeptides (Drosophila capa-1, capa-2, and Manduca sexta CAP2b) raise both [Ca2+]i and cGMP in principal cells (20). Activated NO signaling, however, does not desensitize CAP2b-induced fluid transport (Fig. 4, A and B), whereas overexpression of NOS does not impact on CAP2b-induced [Ca2+]i increases (Figs. 6 and 7). By contrast, treatment of hs dN1-2 tubules with Zaprinast does, however, potentiate the CAP2b-induced secondary [Ca2+]i rise. Thus the secondary calcium rise elicited by CAP2b is dependent on cGMP.

Previous work showed that cGMP induces a slow rise in principal cell [Ca2+]i levels that is dependent on extracellular calcium (30). Expression of the Drosophila gene encoding a cyclic nucleotide-gated channel, cng, has been demonstrated in tubules (30). Drosophila CNG has most similarity to vertebrate CNG3, expressed in nonsensory tissue including kidney (4). We have previously demonstrated that the calcium channel blocker, verapamil, binds to apical membranes in tubule main segment (30). Furthermore, cGMP-induced [Ca2+]i increase and fluid transport are sensitive to verapamil (30), which has also been used as a CNG channel inhibitor in renal blood vessels (26); as such, these responses may occur via CNG channels.

Data presented here suggest that endogenous manipulation of cGMP levels via dNOS transgene expression is insufficient to impact on cGMP-induced [Ca2+]i increases (Figs. 6 and 7). However, use of Zaprinast potentiates cGMP-induced [Ca2+]i increases (Fig. 7, B and D).

We show here that Zaprinast-induced transport phenotype in hs dN1-2 tubules is abolished by verapamil, supporting the idea that the accumulation of high concentrations of cGMP at the apical surface activates calcium channels localized in this region. As such, a major role of cGMP in tubules may be to activate CNG channels. However, given that cGMP has also been shown to activate an apical lumen-positive potential (12), possible, as yet undiscovered, mechanisms may exist to link calcium signaling and V-ATPase action.

In summary, the use of an inducible NOS transgene has revealed a pivotal role of cG-PDE in a transporting epithelium where NO acts as an autocrine signaling molecule.


    APPENDIX
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Summary of the crossing scheme to generate flies homozygous for each of the heat shock GAL4 drivers on chromosome 3 (hsGAL4), the heat shock-driven dNOS transgene (dN1-2) on chromosome 2, and the UAS-apoaequorin reporter line on the X chromosome (UAS:aeq). Other markers used: CyO, the Curly of Oster balancer chromosome, which prevents recombination on chromosome 2, carries a curly wing dominant visible marker, and is homozygous lethal; TM6, a balancer chromosome for chromosome 3, carrying a dominant "tubby" phenotype and which is homozygous lethal; Bl, Bristle, a dominant mutation on chromosome 2, carrying a dominant stubbly bristle phenotype and which is homozygous lethal; TM2, a balancer chromosome for chromosome 3, carrying a dominant pigmented ebony phenotype and which is homozygous lethal; and Y, the Y chromosome.Go



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    DISCLOSURES
 
This work was supported by a Biotechnology and Biological Sciences Research Council (UK) grant (to S. Davies and J. A. T. Dow) and Fellowship support (to S. Davies).


    ACKNOWLEDGMENTS
 
We thank A. K. Allan for helpful discussion, J. McGettigan for development of the NADPH diaphorase assay for Drosophila tubules, R. J. McLennan and L. S. Torrie for help with immunocytochemistry, and N. J. Pyne (University of Strathclyde) for advice and reagents for PDE assays.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. A. Davies, Institute of Biomedical and Life Sciences, Div. of Molecular Genetics, Univ. of Glasgow, Glasgow G11 6NU UK (E-mail: s.a.davies{at}bio.gla.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* K. E. Broderick and M. R. MacPherson contributed equally to this work. Back


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