Drosophila melanogaster NEP2 is a new soluble member of the neprilysin family of endopeptidases with implications for reproduction and renal function.
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The mammalian neprilysin (NEP) family members are typically type II membrane endopeptidases responsible for the activation/inactivation of neuropeptides and peptide hormones. Differences in substrate specificity and subcellular localization of the seven mammalian NEPs contribute to their functional diversity. The sequencing of the Drosophila melanogaster genome has revealed a large expansion of this gene family, resulting in over 20 fly NEP-like genes, suggesting even greater diversity in structure and function than seen in mammals. We now report that one of these genes (Nep2) codes for a secreted endopeptidase with a highly restricted pattern of expression. D. melanogaster NEP2 is expressed in the specialized stellate cells of the renal tubules and in the cyst cells that surround the elongating spermatid bundles in adult testis, suggesting roles for the peptidase in renal function and in spermatogenesis. D. melanogaster NEP2 was found in vesicle-like structures in the syncytial cytoplasm of the spermatid bundles, suggesting that the protein was acquired by endocytosis of protein secreted from the cyst cells. Expression of NEP2 cDNA in D. melanogaster S2 cells confirmed that the peptidase is secreted and is only weakly inhibited by thiorphan, a potent inhibitor of human NEP. D. melanogaster NEP2 also differs from human NEP in the manner in which the peptidase cleaves the tachykinin, GPSGFYGVR-amide. Molecular modelling suggests that there are important structural differences between D. melanogaster NEP2 and human NEP in the S1' and S2' ligand-binding subsites, which might explain the observed differences in inhibitor and substrate specificities. A soluble isoform of a mouse NEP-like peptidase is strongly expressed in spermatids, suggesting an evolutionarily conserved role for a soluble endopeptidase in spermatogenesis. (+info)
Ion-selective microelectrode analysis of salicylate transport by the Malpighian tubules and gut of Drosophila melanogaster.
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Transport of the organic anion salicylate by the Malpighian tubules and gut of larval and adult fruit flies was studied using two salicylate-selective microelectrode methods. The first method combined the high selectivity of tridodecylmethylammonium-based electrodes for salicylate with the self-referencing ion-selective microelectrode technique for non-invasive spatial and temporal analysis of salicylate flux. Measurements with this technique revealed secretion of salicylate across the main and distal segments of the Malpighian tubule as well as the midgut, ileum and rectum. The second method used a salicylate-selective microelectrode to measure the concentration of salicylate in fluid droplets secreted by isolated Drosophila Malpighian tubules set up in a Ramsay secretion assay. Transepithelial salicylate flux was calculated as the product of fluid secretion rate and secreted fluid salicylate concentration. Measurements with this method revealed that salicylate transport was active and saturable; the kinetic parameters J(max) and K(t) were 2.72 pmol min(-1) tubule(-1) and 0.046 mmol l(-1), respectively. Measurements of transepithelial salicylate flux determined by both microelectrode methods were in good agreement. Transepithelial flux measurements measured by microelectrodes were also validated by comparing them with measurements of radiolabelled salicylate levels in secreted droplets. Salicylate concentrations in haemolymph samples were measured with salicylate-selective microelectrodes after injection of salicylate into the haemocoel or after insects were fed salicylate-rich diets. The rate of salicylate secretion by Malpighian tubules in vitro was sufficient to account for the measured rate of decline of salicylate concentration in the haemolymph in vivo. (+info)
Nitric oxide scavenging by the cobalamin precursor cobinamide.
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Nitric oxide (NO) is an important signaling molecule, and a number of NO synthesis inhibitors and scavengers have been developed to allow study of NO functions and to reduce excess NO levels in disease states. We showed previously that cobinamide, a cobalamin (vitamin B12) precursor, binds NO with high affinity, and we now evaluated the potential of cobinamide as a NO scavenger in biologic systems. We found that cobinamide reversed NO-stimulated fluid secretion in Drosophila Malpighian tubules, both when applied in the form of a NO donor and when produced intracellularly by nitricoxide synthase. Moreover, feeding flies cobinamide markedly attenuated subsequent NO-induced increases in tubular fluid secretion. Cobinamide was taken up efficiently by cultured rodent cells and prevented NO-induced phosphorylation of the vasodilator-stimulated phosphoprotein VASP both when NO was provided to the cells and when NO was generated intracellularly. Cobinamide appeared to act via scavenging NO because it reduced nitrite and nitrate concentrations in both the fly and mammalian cell systems, and it did not interfere with cGMP-induced phosphorylation of VASP. In rodent and human cells, cobinamide exhibited toxicity at concentrations > or =50 microM with toxicity completely prevented by providing equimolar amounts of cobalamin. Combining cobalamin with cobinamide had no effect on the ability of cobinamide to scavenge NO. Cobinamide did not inhibit the in vitro activity of either of the two mammalian cobalamin-dependent enzymes, methionine synthase or methylmalonyl-coenzyme A mutase; however, it did inhibit the in vivo activities of the enzymes in the absence, but not presence, of cobalamin, suggesting that cobinamide toxicity was secondary to interference with cobalamin metabolism. As part of these studies, we developed a facile method for producing and purifying cobinamide. We conclude that cobinamide is an effective intra- and extracellular NO scavenger whose modest toxicity can be eliminated by cobalamin. (+info)
Renal tubule development in Drosophila: a closer look at the cellular level.
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The function of excretion in insects is performed by the Malpighian tubules, a functional equivalent of the vertebrate kidney. Malpighian tubules are long, thin tubes connected to the hindgut. Upon the determination of the Malpighian tubule major cell type early in embryogenesis, the tubular architecture is achieved by extensive cell division and cell rearrangements. During the tube elongation process, cells exchange their neighbors, allowing the short and fat Malpighian tubule primordia to grow and become a thin tube. Cell rearrangement and intercalation underlie the morphogenesis of other epithelial tissues in Drosophila melanogaster, such as the embryonic epidermis. Recent work has provided insights in the cellular and molecular basis of cell intercalation. These advances are reviewed and discussed with regard to what is known about Malpighian tubule morphogenesis. Mature Malpighian tubules are composed of two cell types, each having a specific function in excretion: The principal cells and the stellate cells. Drosophila and mammalian kidney development show striking similarities, as the recruitment of the stellate cells to the Malpighian tubules, like the cells of the metanephric mesenchyme, requires that cells undergo a mesenchymal-to-epithelial transition. The molecular similarities between these two cases is reviewed here. (+info)
Transient receptor potential-like channels are essential for calcium signaling and fluid transport in a Drosophila epithelium.
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Calcium signaling is an important mediator of neuropeptide-stimulated fluid transport by Drosophila Malpighian (renal) tubules. We demonstrate the first epithelial role, in vivo, for members of the TRP family of calcium channels. RT-PCR revealed expression of trp, trpl, and trpgamma in tubules. Use of antipeptide polyclonal antibodies for TRP, TRPL, and TRPgamma showed expression of all three channels in type 1 (principal) cells in the tubule main segment. Neuropeptide (CAP(2b))-stimulated fluid transport rates were significantly reduced in tubules from the trpl(302) mutant and the trpl;trp double mutant, trpl(302);trp(343). However, a trp null, trp(343), had no impact on stimulated fluid transport. Measurement of cytosolic calcium concentrations ([Ca(2+)](i)) in tubule principal cells using an aequorin transgene in trp and trpl mutants showed a reduction in calcium responses in trpl(302). Western blotting of tubule preparations from trp and trpl mutants revealed a correlation between TRPL levels and CAP(2b)-stimulated fluid transport and calcium signaling. Rescue of trpl(302) with a trpl transgene under heat-shock control resulted in a stimulated fluid transport phenotype that was indistinguishable from wild-type tubules. Furthermore, restoration of normal stimulated rates of fluid transport by rescue of trpl(302) was not compromised by introduction of the trp null, trp(343). Thus, in an epithelial context, TRPL is sufficient for wild-type responses. Finally, a scaffolding component of the TRPL/TRP-signaling complex, INAD, is not expressed in tubules, suggesting that inaD is not essential for TRPL/TRP function in Drosophila tubules. (+info)
Making tubes in the Drosophila embryo.
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Epithelial and endothelial tubes come in various shapes and sizes and form the basic units of many tubular organs. During embryonic development, single unbranched tubes as well as highly branched networks of tubes form from simple sheets of cells by several morphogenic movements. Studies of tube formation in the Drosophila embryo have greatly advanced our understanding of the cellular and molecular mechanisms by which tubes are formed. This review highlights recent progress on formation of the hindgut, Malpighian tubules, proventriculus, salivary gland, and trachea of the Drosophila embryo, focusing on the cellular events that form each tube and their genetic requirements. (+info)
A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling.
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The Drosophila orphan G protein-coupled receptor encoded by CG17415 is related to members of the calcitonin receptor-like receptor (CLR) family. In mammals, signaling from CLR receptors depend on accessory proteins, namely the receptor activity modifying proteins (RAMPs) and receptor component protein (RCP). We tested the possibility that this Drosophila CLR might also require accessory proteins for proper function and we report that co-expression of the mammalian or Drosophila RCP or mammalian RAMPs permitted neuropeptide diuretic hormone 31 (DH31) signaling from the CG17415 receptor. RAMP subtype expression did not alter the pharmacological profile of CG17415 activation. CG17415 antibodies revealed expression within the principal cells of Malpighian tubules, further implicating DH31 as a ligand for this receptor. Immunostaining in the brain revealed an unexpected convergence of two distinct DH signaling pathways. In both the larval and adult brain, most DH31 receptor-expressing neurons produce the neuropeptide corazonin, and also express the CRFR-related receptor CG8422, which is a receptor for the neuropeptide diuretic hormone 44 (DH44). There is extensive convergence of CRF and CGRP signaling within vertebrates and we report a striking parallel in Drosophila involving DH44 (CRF) and DH31 (CGRP). Therefore, it appears that both the molecular details as well as the functional organization of CGRP signaling have been conserved. (+info)
Developmental expression and biophysical characterization of a Drosophila melanogaster aquaporin.
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Aquaporins (AQPs) accelerate the movement of water and other solutes across biological membranes, yet the molecular mechanisms of each AQP's transport function and the diverse physiological roles played by AQP family members are still being defined. We therefore have characterized an AQP in a model organism, Drosophila melanogaster, which is amenable to genetic manipulation and developmental analysis. To study the mechanism of Drosophila Malpighian tubule (MT)-facilitated water transport, we identified seven putative AQPs in the Drosophila genome and found that one of these, previously named DRIP, has the greatest sequence similarity to those vertebrate AQPs that exhibit the highest rates of water transport. In situ mRNA analyses showed that DRIP is expressed in both embryonic and adult MTs, as well as in other tissues in which fluid transport is essential. In addition, the pattern of DRIP expression was dynamic. To define DRIP-mediated water transport, the protein was expressed in Xenopus oocytes and in yeast secretory vesicles, and we found that significantly elevated rates of water transport correlated with DRIP expression. Moreover, the activation energy required for water transport in DRIP-expressing secretory vesicles was 4.9 kcal/mol. This low value is characteristic of AQP-mediated water transport, whereas the value in control vesicles was 16.4 kcal/mol. In contrast, glycerol, urea, ammonia, and proton transport were unaffected by DRIP expression, suggesting that DRIP is a highly selective water-specific channel. This result is consistent with the homology between DRIP and mammalian water-specific AQPs. Together, these data establish Drosophila as a new model system with which to investigate AQP function. (+info)