The mechanism of action of the antidiuretic peptide Tenmo ADFa in Malpighian tubules of Aedes aegypti.
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The mechanism of action of Tenebrio molitor antidiuretic factor 'a' (Tenmo ADFa) was explored in isolated Malpighian tubules of Aedes aegypti. In the Ramsay assay of fluid secretion, Tenmo ADFa (10(-9) mol l(-1)) significantly inhibited the rate of fluid secretion from 0.94 nl min(-1) to 0.44 nl min(-1) without significant effects on the concentrations of Na+, K+ and Cl- in secreted fluid. In isolated perfused tubules, Tenmo ADFa had no effect on the transepithelial voltage (Vt) and resistance (Rt). In principal cells of the tubule, Tenmo ADFa had no effect on the basolateral membrane voltage (Vbl) and the input resistance of principal cells (Rpc). Tenmo ADFa significantly increased the intracellular concentration of cyclic guanosine monophosphate (cGMP) from 2.9 micromol l(-1) (control) to 7.4 micromol l(-1). A peritubular [cGMP] of 20 micromol l(-1) duplicated the antidiuretic effects of Tenmo ADFa without inducing electrophysiological effects. In contrast, 500 micromol l(-1) cGMP significantly depolarized V(bl), hyperpolarized Vt, and reduced Rt and Rpc, without increasing antidiuretic potency beyond that of 20 micromol l(-1) cGMP. A plot of peritubular cGMP concentration vs Vbl revealed a steep dose-response between 300 micromol l(-1) and 700 micromol l(-1) with an EC50 of 468 micromol l(-1). These observations suggest a receptor- and cGMP-mediated mechanism of action of Tenmo ADFa. Tenmo ADFa and physiological concentrations of cGMP (< 20 micromol l(-1)) reduce the rate of isosmotic fluid secretion by quenching electroneutral transport systems. The inhibition reveals that as much as 50% of the normal secretory solute and water flux can stem from electrically silent mechanisms in this highly electrogenic epithelium. (+info)
Cell-specific manipulation of second messengers; a toolbox for integrative physiology in Drosophila.
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Every living cell must detect, and respond appropriately to, external signals. The functions of intracellular second messengers, such as guanosine 3'5'-cyclic monophosphate (cGMP), adenosine 3'5'-cyclic monophosphate (cAMP), and intracellular calcium, are thus intensively studied. However, artifact-free manipulation of these messengers is problematic, and simple pharmacology may not allow selective intervention in distinct cell types in a real, complex tissue. We have devised a method by which second messenger levels can be manipulated in cells of choice using the GAL4/UAS system. By placing different receptors (rat atrial natriuretic peptide [ANP] receptor and Drosophila serotonin receptors [5HT(Dro7) and 5HT(Dro1A)]) under UAS control, they can be targeted to arbitrary defined populations of cells in any tissue of the fly, and second messenger levels can be manipulated simply by adding the natural ligand. The potential of the system is illustrated in the Drosophila renal (Malpighian) tubule, where each receptor was shown to stimulate fluid secretion, to act through its cognate second messenger, and to be blocked by appropriate pharmacological antagonists. The results uncovered a new role for cGMP signaling in tubule and also demonstrate the utility of the tubule as a possible in vivo test bed for novel receptors, ligands, or agonists/antagonists. (+info)
A conserved domain of alkaline phosphatase expression in the Malpighian tubules of dipteran insects.
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Malpighian (renal) tubules are key components of the insect osmoregulatory system and show correspondingly great diversity in both number and length. Recently, the organisation of the Drosophila melanogaster tubule has been elucidated by enhancer trapping, and an array for functional properties has been shown to align with the functional domains. In Drosophila, there is a lower tubule domain, which coincides with expression of alkaline phosphatase and delineates the absorptive region of the tubule. Here, these observations are extended to three dipteran vectors of disease (Aedes aegypti, Anopheles stephensii and Glossina morsitans) and a non-dipteran out-group, Schistocerca gregaria (Orthoptera). Despite a huge range in cell number and size, alkaline phosphatase was found on the apical surface of the lower 10% of each of the dipteran tubules but nowhere within the orthopteran tubule. An alkaline phosphatase lower tubule domain is thus conserved among Diptera. Cell counts are also provided for each species. As in Drosophila, stellate cells are not found in the lower tubule domain of Anopheles or Aedes tubules, confirming the unique genetic identity of this domain. As previously reported, we failed to find stellate cells in Schistocerca but, remarkably, also failed to find them in Glossina, the dipteran most closely related to Drosophila. The orthodoxy that stellate cells are unique to, and general among, Diptera may thus require revision. (+info)
Function-informed transcriptome analysis of Drosophila renal tubule.
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BACKGROUND: Comprehensive, tissue-specific, microarray analysis is a potent tool for the identification of tightly defined expression patterns that might be missed in whole-organism scans. We applied such an analysis to Drosophila melanogaster Malpighian (renal) tubule, a defined differentiated tissue. RESULTS: The transcriptome of the D. melanogaster Malpighian tubule is highly reproducible and significantly different from that obtained from whole-organism arrays. More than 200 genes are more than 10-fold enriched and over 1,000 are significantly enriched. Of the top 200 genes, only 18 have previously been named, and only 45% have even estimates of function. In addition, 30 transcription factors, not previously implicated in tubule development, are shown to be enriched in adult tubule, and their expression patterns respect precisely the domains and cell types previously identified by enhancer trapping. Of Drosophila genes with close human disease homologs, 50 are enriched threefold or more, and eight enriched 10-fold or more, in tubule. Intriguingly, several of these diseases have human renal phenotypes, implying close conservation of renal function across 400 million years of divergent evolution. CONCLUSIONS: From those genes that are identifiable, a radically new view of the function of the tubule, emphasizing solute transport rather than fluid secretion, can be obtained. The results illustrate the phenotype gap: historically, the effort expended on a model organism has tended to concentrate on a relatively small set of processes, rather than on the spread of genes in the genome. (+info)
Resolution of the insect ouabain paradox.
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Many insects are highly resistant to plant toxins, such as the cardiac glycoside ouabain. How can the epithelia that must handle such toxins, also be refractory to them? In Drosophila, the Malpighian (renal) tubule contains large amounts of Na(+),K(+) ATPase that is known biochemically to be exquisitely sensitive to ouabain, yet the intact tissue is almost unaffected by even extraordinary concentrations. The explanation is that the tubules are protected by an active ouabain transport system, colocated with the Na(+),K(+) ATPase, thus preventing ouabain from reaching inhibitory concentrations within the basolateral infoldings of principal cells. These data show that the Na(+),K(+) ATPase, previously thought to be unimportant, may be as vital in insect tissues as in vertebrates, but can be cryptic to conventional pharmacology. (+info)
Antibodies to mammalian and plant V-ATPases cross react with the V-ATPase of insect cation-transporting plasma membranes.
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In immunobiochemical blots, polyclonal antibodies against subunits of plant and mammalian vacuolar-type ATPases (V-ATPases) cross-react strongly with corresponding subunits of larval Manduca sexta midgut plasma membrane V-ATPase. Thus, rabbit antiserum against Kalanchoe daigremontiana tonoplast V-ATPase holoenzyme cross-reacts with the 67, 56, 40, 28 and 20 kDa subunits of midgut V-ATPase separated by SDS-PAGE. Antisera against bovine chromaffin granule 72 and 39 kDa V-ATPase subunits cross-react with the corresponding 67 and 43 kDa subunits of midgut V-ATPase. Antisera against the 57 kDa subunit of both beet root and oat root V-ATPase cross-react strongly with the midgut 56 kDa V-ATPase subunit. In immunocytochemical light micrographs, antiserum against the beet root 57 kDa V-ATPase subunit labels the goblet cell apical membrane of both posterior and anterior midgut in freeze-substituted and fixed sections. The plant antiserum also labels the apical brush-border plasma membrane of Malpighian tubules. The ability of antibodies against plant V-ATPase to label these insect membranes suggests a high sequence homology between V-ATPases from plants and insects. Both of the antibody-labelled insect membranes transport K+ and both membranes possess F1-like particles, portasomes, on their cytoplasmic surfaces. This immunolabelling by xenic V-ATPase antisera of two insect cation-transporting membranes suggests that the portasomes on these membranes may be V-ATPase particles, similar to those reported on V-ATPase-containing vacuolar membranes from various sources. (+info)
Na+ competes with K+ in bumetanide-sensitive transport by Malpighian tubules of Rhodnius prolixus.
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We examined the effects of bathing saline Na+/K+ ratio, bumetanide and hydrochlorothiazide on fluid and ion transport by serotonin-stimulated Malpighian tubules of Rhodnius prolixus. Previous pharmacological and electrophysiological studies indicate that a bumetanide-sensitive Na+/K+/2Cl- cotransporter is the primary route for basolateral ion entry into the cell during fluid secretion. The goal of this study was to resolve the apparent conflict between relatively high secretion rates by tubules bathed in K+-free saline and the evidence that Na+/K+/2Cl- cotransporters described in other systems have an absolute requirement for all three ions for translocation. Our measurements of fluid secretion rate, ion fluxes and electrophysiological responses to serotonin show that fluid secretion in K+-free saline is bumetanide sensitive and hydrochlorothiazide insensitive. Dose-response curves of secretion rate versus bumetanide concentration were identical for tubules bathed in K+-free and control saline with IC50 values of 2.6 x 10(-6) mmol l(-1) and 2.9 x 10(-6) mmol l(-1), respectively. Double-reciprocal plots of K+ flux versus bathing saline K+ concentration showed that increasing Na+ concentration in the bathing fluid increased Kt but had no effect on Jmax, consistent with competitive inhibition of K+ transport by Na+. We propose that the competition between Na+ and K+ for transport by the bumetanide-sensitive transporter is part of an autonomous mechanism by which Malpighian tubules regulate haemolymph K+ concentration. (+info)
Conservation of capa peptide-induced nitric oxide signalling in Diptera.
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In D. melanogaster Malpighian (renal) tubules, the capa peptides stimulate production of nitric oxide (NO) and guanosine 3', 5'-cyclic monophosphate (cGMP), resulting in increased fluid transport. The roles of NO synthase (NOS), NO and cGMP in capa peptide signalling were tested in several other insect species of medical relevance within the Diptera (Aedes aegypti, Anopheles stephensi and Glossina morsitans) and in one orthopteran out-group, Schistocerca gregaria. NOS immunoreactivity was detectable by immunocytochemistry in tubules from all species studied. D. melanogaster, A. aegypti and A. stephensi express NOS in only principal cells, whereas G. morsitans and S. gregaria show more general NOS expression in the tubule. Measurement of associated NOS activity (NADPH diaphorase) shows that both D. melanogaster capa-1 and the two capa peptides encoded in the A. gambiae genome, QGLVPFPRVamide (AngCAPA-QGL) and GPTVGLFAFPRVamide (AngCAPA-GPT), all stimulate NOS activity in D. melanogaster, A. aegypti, A. stephensi and G. morsitans tubules but not in S. gregaria. Furthermore, capa-stimulated NOS activity in all the Diptera was inhibited by the NOS inhibitor l-NAME. All capa peptides stimulate an increase in cGMP content across the dipteran species, but not in the orthopteran S. gregaria. Similarly, all capa peptides tested stimulate fluid secretion in D. melanogaster, A. aegypti, A. stephensi and G. morsitans tubules but are either without effect or are inhibitory on S. gregaria. Consistent with these results, the Drosophila capa receptor was shown to be expressed in Drosophila tubules, and its closest Anopheles homologue was shown to be expressed in Anopheles tubules. Thus, we provide the first demonstration of physiological roles for two putative A. gambiae neuropeptides. We also demonstrate neuropeptide modulation of fluid secretion in tsetse tubule for the first time. Finally, we show the generality of capa peptide action, to stimulate NO/cGMP signalling and increase fluid transport, across the Diptera, but not in the more primitive Orthoptera. (+info)