RESUMO
Cold-acclimated insects defend ion and water transport function during cold exposure. We hypothesized that this is achieved via enhanced active transport. The Malpighian tubules and rectum are likely targets for such transport modifications, and recent transcriptomic studies indicate shifts in Na+-K+ ATPase (NKA) and V-ATPase expression in these tissues following cold acclimation. Here we quantify the effect of cold acclimation (one week at 12°C) on active transport in the ionoregulatory organs of adult Gryllus pennsylvanicus field crickets. We compared primary urine production of warm- and cold-acclimated crickets in excised Malpighian tubules via Ramsay assay at a range of temperatures between 4 and 25°C. We then compared NKA and V-ATPase activities in Malpighian tubule and rectal homogenates from warm- and cold-acclimated crickets via NADH-linked photometric assays. Malpighian tubules of cold-acclimated crickets excreted fluid at lower rates at all temperatures compared to warm-acclimated crickets. This reduction in Malpighian tubule excretion rates may be attributed to increased NKA activity that we observed for cold-acclimated crickets, but V-ATPase activity was unchanged. Cold acclimation had no effect on rectal NKA activity at either 21°C or 6°C, and did not modify rectal V-ATPase activity. Our results suggest that an overall reduction, rather than enhancement of active transport in the Malpighian tubules allows crickets to maintain hemolymph water balance during cold exposure, and increased Malpighian tubule NKA activity may help to defend and/or re-establish ion homeostasis.
Assuntos
Aclimatação , Temperatura Baixa , Gryllidae/fisiologia , Transporte de Íons , Animais , Túbulos de Malpighi/enzimologia , Túbulos de Malpighi/metabolismo , Reto/enzimologia , Reto/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Transcriptoma , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Spodoptera frugiperda is a polyphagous lepidopteran pest that encounters a wide range of toxic plant metabolites in its diet. The ability of this insect to adapt to its chemical environment might be explained by the action of major detoxification enzymes such as cytochrome P450s (or CYP). Forty-two sequences coding for P450s were identified and most of the transcripts were found to be expressed in the midgut, Malpighian tubules and fat body of S. frugiperda larvae. Relatively few P450s were expressed in the established cell line Sf9. In order to gain information on how these genes respond to different chemical compounds, larvae and Sf9 cells were exposed to plant secondary metabolites (indole, indole-3-carbinol, quercetin, 2-tridecanone and xanthotoxin), insecticides (deltamethrin, fipronil, methoprene, methoxyfenozide) or model inducers (clofibrate and phenobarbital). Several genes were induced by plant chemicals such as P450s from the 6B, 321A and 9A subfamilies. Only a few genes responded to insecticides, belonging principally to the CYP9A family. There was little overlap between the response in vivo measured in the midgut and the response in vitro in Sf9 cells. In addition, regulatory elements were detected in the promoter region of these genes. In conclusion, several P450s were identified that could potentially be involved in the adaptation of S. frugiperda to its chemical environment.
Assuntos
Indutores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Inseticidas/farmacologia , Feromônios/farmacologia , Spodoptera/enzimologia , Spodoptera/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Sistema Digestório/enzimologia , Corpo Adiposo/enzimologia , Perfilação da Expressão Gênica , Larva/enzimologia , Túbulos de Malpighi/enzimologia , Dados de Sequência Molecular , Plantas/química , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Malpighian tubules are the osmoregulatory and detoxifying organs of Drosophila and its proper development is critical for the survival of the organism. They are made up of two major cell types, the ectodermal principal cells and mesodermal stellate cells. The principal and stellate cells are structurally and physiologically distinct from each other, but coordinate together for production of isotonic fluid. Proper integration of these cells during the course of development is an important pre-requisite for the proper functioning of the tubules. We have conclusively determined an essential role of ecdysone hormone in the development and function of Malpighian tubules. Disruption of ecdysone signaling interferes with the organization of principal and stellate cells resulting in malformed tubules and early larval lethality. Abnormalities include reduction in the number of cells and the clustering of cells rather than their arrangement in characteristic wild type pattern. Organization of F-actin and ß-tubulin also show aberrant distribution pattern. Malformed tubules show reduced uric acid deposition and altered expression of Na(+)/K(+)-ATPase pump. B2 isoform of ecdysone receptor is critical for the development of Malpighian tubules and is expressed from early stages of its development.
Assuntos
Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Ecdisona/farmacologia , Túbulos de Malpighi/embriologia , Morfogênese/efeitos dos fármacos , Receptores de Esteroides/metabolismo , Actinas/metabolismo , Animais , Contagem de Células , Citoesqueleto/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Larva/efeitos dos fármacos , Larva/metabolismo , Túbulos de Malpighi/anormalidades , Túbulos de Malpighi/enzimologia , Túbulos de Malpighi/patologia , Mutação , Fenótipo , Isoformas de Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Tubulina (Proteína)/metabolismo , Ácido Úrico/metabolismoRESUMO
Transepithelial ion transport in insect Malpighian tubules is energized by an apical V-ATPase. In hematophagous insects, a blood meal during which the animal ingests huge amounts of salt and water stimulates transepithelial transport processes linked to V-ATPase activation, but how this is accomplished is still unclear. Here we report that membrane-permeant derivatives of cAMP increase the bafilomycin-sensitive ATPase activity in Malpighian tubules of Aedes aegypti twofold and activate ATP-dependent transport processes. In parallel, membrane association of the V(1) subunits C and D increases, consistent with the assembly of the holoenzyme. The protein kinase A inhibitor H-89 abolishes all cAMP-induced effects, consistent with protein kinase A (PKA) being involved in V-ATPase activation. Metabolic inhibition induced by KCN, azide and 2,4-dinitrophenol, respectively, also induces assembly of functional V-ATPases at the membrane without PKA involvement, indicating a phosphorylation-independent activation mechanism.
Assuntos
Aedes/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Túbulos de Malpighi/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Diurese , Feminino , Túbulos de Malpighi/metabolismo , Serotonina/metabolismoRESUMO
Multicopper ferroxidases catalyze the oxidation of ferrous iron to ferric iron. In yeast and algae, they participate in cellular uptake of iron; in mammals, they facilitate cellular efflux. The mechanisms of iron metabolism in insects are still poorly understood, and insect multicopper ferroxidases have not been identified. In this paper, we present evidence that Drosophila melanogaster multicopper oxidase-1 (MCO1) is a functional ferroxidase. We identified candidate iron-binding residues in the MCO1 sequence and found that purified recombinant MCO1 oxidizes ferrous iron. An association between MCO1 function and iron homeostasis was confirmed by two observations: RNAi-mediated knockdown of MCO1 resulted in decreased iron accumulation in midguts and whole insects, and weak knockdown increased the longevity of flies fed a toxic concentration of iron. Strong knockdown of MCO1 resulted in pupal lethality, indicating that MCO1 is an essential gene. Immunohistochemistry experiments demonstrated that MCO1 is located on the basal surfaces of the digestive system and Malpighian tubules. We propose that MCO1 oxidizes ferrous iron in the hemolymph and that the resulting ferric iron is bound by transferrin or melanotransferrin, leading to iron storage, iron withholding from pathogens, regulation of oxidative stress, and/or epithelial maturation. These proposed functions are distinct from those of other known ferroxidases. Given that MCO1 orthologues are present in all insect genomes analyzed to date, this discovery is an important step toward understanding iron metabolism in insects.
Assuntos
Ceruloplasmina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Homeostase , Ferro/metabolismo , Oxirredutases/metabolismo , Aminoácidos/metabolismo , Animais , Sítios de Ligação , Biocatálise/efeitos dos fármacos , Sistema Digestório/citologia , Sistema Digestório/enzimologia , Drosophila melanogaster/citologia , Drosophila melanogaster/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Homeostase/efeitos dos fármacos , Imuno-Histoquímica , Ferro/farmacologia , Lacase/metabolismo , Longevidade/efeitos dos fármacos , Masculino , Túbulos de Malpighi/citologia , Túbulos de Malpighi/enzimologia , Homologia de Sequência de AminoácidosRESUMO
Mg(2+)-dependent ATPases were investigated in Malpighian tubules of the blood-sucking insect, Triatoma infestans, with cytochemical procedures for light and electron microscopy. The aim was to establish patterns of enzyme occurrence in the blood-sucking insect under control rearing conditions for further comparisons with animals subjected to the action of stress factors. Enzyme activity was found in laminated "concretions" present in distal cells, in edges of urate crystals at the lumen of the proximal region of tubules, in the basement membrane of proximal cells, and variously distributed in plasmalemma invaginations of both distal and proximal cells. Presence of ATPases in the "concretions" and urate crystals is presumed to be due to engulfment of other ATPase-containing components during formation of these structures. Cytochemical reactivity in the basement membrane and plasmalemma invaginations is assumed to be involved with active transport of waste molecules from and to hemolymph and differs as a function of the Malpighian tubule region. This paper provides a basic understanding of the enzyme occurrence in the blood sucking insects, and can be used as a pattern for comparative means of the staining patterns among Triatominae species.
Assuntos
Adenosina Trifosfatases/metabolismo , Cátions Bivalentes/metabolismo , Magnésio/metabolismo , Triatoma/enzimologia , Animais , Histocitoquímica , Túbulos de Malpighi/enzimologia , MicroscopiaRESUMO
Organismal stress responses to oxidative stress are relevant to ageing and disease and involve key cell-/tissue-specific signal transduction mechanisms. Using Drosophila, an established in vivo model for stress studies, we show that cell-specific inositol phosphate signalling specifically via inositol 1,4,5 trisphosphate 3-kinase (InsP(3) 3-K, IP(3)K), negatively regulates organismal responses to oxidative stress. We demonstrate that the Drosophila Malpighian tubule (equivalent to vertebrate kidney and liver) is a key epithelial sensor for organismal oxidative stress responses: precise targeting of either gain-of-function constructs of Drosophila IP(3)Ks (IP(3)K-1 and IP(3)K-2), or loss-of-function (RNAi) constructs to only one cell type in tubule reversibly modulates survival of stress-challenged adult flies. In vivo, targeted IP(3)K-1 directly increases H(2)O(2) production, pro-apoptotic caspase-9 activity and mitochondrial membrane potential. The mitochondrial calcium load in tubule principal cells-assessed by luminescent and fluorescent genetically-encoded mitochondrial calcium reporters-is significantly increased by IP(3)K-1 under oxidative stress conditions, leading to apoptosis. The Drosophila orthologues of human apoptotic bcl-2 genes include debcl and buffy. Oxidative stress challenge does not modulate gene expression of either debcl or buffy in tubules; and altered debcl expression does not influence survival rates under oxidative stress challenge. Finally, targeted over-expression of either debcl or buffy to tubule principal cells does not impact on tubule caspase-9 activity. Thus, IP(3)K-1 modulates epithelial cell apoptosis without involvement of bcl-2-type proteins.
Assuntos
Apoptose , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Estresse Oxidativo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Cálcio/metabolismo , Caspase 9/metabolismo , Peróxido de Hidrogênio/metabolismo , Túbulos de Malpighi/citologia , Túbulos de Malpighi/enzimologia , Potencial da Membrana Mitocondrial , Mitocôndrias/enzimologia , Especificidade de Órgãos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Análise de SobrevidaRESUMO
Mammalian nucleostemin (NS) is a nucleolar guanosine triphosphate-binding protein implicated in cell cycle progression, stem cell proliferation, and ribosome assembly. Drosophila melanogaster contains a four-member nucleostemin family (NS1-4). NS1 is the closest orthologue to human NS; it shares 33% identity and 67% similarity with human NS. We show that NS1 has intrinsic GTPase and ATPase activity and that it is present within nucleoli of most larval and adult cells. Endogenous NS1 and lightly expressed green fluorescent protein (GFP)-NS1 enrich within the nucleolar granular regions as expected, whereas overexpressed GFP-NS1 localized throughout the nucleolus and nucleoplasm, and to several transcriptionally active interbands of polytene chromosomes. Severe overexpression correlated with the appearance of melanotic tumors and larval/pupal lethality. Depletion of 60% of NS1 transcripts also lead to larval and pupal lethality. NS1 protein depletion>95 correlated with the loss of imaginal island (precursor) cells in the larval midgut and to an apparent block in the nucleolar release of large ribosomal subunits in terminally differentiated larval midgut polyploid cells. Ultrastructural examination of larval Malpighian tubule cells depleted for NS1 showed a loss of cytoplasmic ribosomes and a concomitant appearance of cytoplasmic preautophagosomes and lysosomes. We interpret the appearance of these structures as indicators of cell stress response.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Drosophila/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Guanosina Trifosfato/metabolismo , Intestinos/citologia , Subunidades Ribossômicas Maiores/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Nucléolo Celular/enzimologia , Cromossomos/ultraestrutura , Sequência Conservada , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Ligação ao GTP/deficiência , Proteínas de Ligação ao GTP/genética , Deleção de Genes , Técnicas de Silenciamento de Genes , Genes Reporter , Intestinos/enzimologia , Intestinos/crescimento & desenvolvimento , Larva , Lisossomos/fisiologia , Túbulos de Malpighi/enzimologia , Túbulos de Malpighi/ultraestrutura , Dados de Sequência Molecular , Neoplasias Experimentais/genética , Fagossomos/fisiologia , Pupa , Interferência de RNA , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Given the physiological importance of the Malpighian tubules to homeostasis in ants, this study aimed to characterize the enzymology, histology, histochemistry, and ultramorphology of the Malpighian tubules of Cephalotes atratus, C. clypeatus, and C. pusillus, as a contribution for the understanding of this organ, as well as to examine its role in the maintenance of symbiontic microorganisms in the ileum of these ants.
Assuntos
Formigas/microbiologia , Túbulos de Malpighi/microbiologia , Simbiose , Adenosina Trifosfatases/análise , Animais , Concentração de Íons de Hidrogênio , Túbulos de Malpighi/enzimologia , Túbulos de Malpighi/ultraestruturaRESUMO
This study describes the expression patterns of P-type Na(+)/K(+)-ATPase and V-type H(+)-ATPase in the larval and adult forms of the mosquito Aedes aegypti and provides insight into their relative importance in ion transport function of key osmoregulatory organs. RT-PCR assays indicate that, at the level of the gene, both ATPases are expressed in all of the osmoregulatory tissues of larvae (midgut, Malpighian tubules, rectum and anal papillae) and adults (stomach, Malpighian tubules, anterior hindgut and rectum). Immunohistochemical studies determined that both ATPases are present in high levels in all the relevant organs, with the exception of the larval rectum (P-type Na(+)/K(+)-ATPase only). In larval gastric caeca, ATPase location corresponds to the secretory (basal P-type Na(+)/K(+)-ATPase, apical V-type H(+)-ATPase) and ion-transporting (V-type H(+)-ATPase on both membranes) regions as previously described. The two ATPases switch membrane location along the length of the larval midgut, indicating three possible regionalizations, whereas the adult stomach has uniform expression of basolateral P-type Na(+)/K(+)-ATPase and apical V-type H(+)-ATPase in each cell. In both larval and adult Malpighian tubules, the distal principal cells exhibit high expression levels of V-type H(+)-ATPase (apically and cytoplasmically) whereas P-type Na(+)/K(+)-ATPase is highly expressed in stellate cells found only in the distal two-thirds of each tubule. By contrast, the proximal principal cells express both P-type Na(+)/K(+)-ATPase (basal) and V-type H(+)-ATPase (apical). These results suggest a functional segregation along the length of the Malpighian tubules based on cell type and region. P-type Na(+)/K(+)-ATPase is the only pump apparent in the larval rectum whereas in the larval anal papillae and the adult hindgut (including the anterior hindgut and rectum with rectal pads), P-type Na(+)/K(+)-ATPase and V-type H(+)-ATPase localize to the basal and apical membranes, respectively. We discuss our findings in light of previous physiological and morphological studies and re-examine our current models of ion transport in these two developmental stages of mosquitoes that cope with disparate osmoregulatory challenges.
Assuntos
Aedes/metabolismo , ATPases Translocadoras de Prótons/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia , Animais , Feminino , Trato Gastrointestinal/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Larva/enzimologia , Túbulos de Malpighi/enzimologiaRESUMO
Secretory pathway Ca2+/Mn2+-ATPases (SPCAs) are important for maintenance of cellular Ca2+ and Mn2+ homeostasis, and, to date, all SPCAs have been found to localize to the Golgi apparatus. The single Drosophila SPCA gene (SPoCk) was identified by an in silico screen for novel Ca2+-ATPases. It encoded three SPoCk isoforms with novel, distinct subcellular specificities in the endoplasmic reticulum (ER) and peroxisomes in addition to the Golgi. Furthermore, expression of the peroxisome-associated SPoCk isoform was sexually dimorphic. Overexpression of organelle-specific SPoCk isoforms impacted on cytosolic Ca2+ handling in both cultured Drosophila cells and a transporting epithelium, the Drosophila Malpighian (renal) tubule. Specifically, the ER isoform impacted on inositol-trisphosphate-mediated Ca2+ signaling and the Golgi isoform impacted on diuresis, whereas the peroxisome isoform colocalized with Ca2+ "spherites" and impacted on calcium storage and transport. Interfering RNA directed against the common exons of the three SPoCk isoforms resulted in aberrant Ca2+ signaling and abolished neuropeptide-stimulated diuresis by the tubule. SPoCk thus contributed to both of the contrasting requirements for Ca2+ in transporting epithelia: to transport or store Ca2+ in bulk without compromising its use as a signal.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Túbulos de Malpighi/enzimologia , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio , ATPases Transportadoras de Cálcio/genética , Linhagem Celular , Diurese , Proteínas de Drosophila/genética , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Retículo Endoplasmático/enzimologia , Complexo de Golgi/enzimologia , Isoenzimas/genética , Isoenzimas/metabolismo , Túbulos de Malpighi/efeitos dos fármacos , Manganês/metabolismo , Peroxissomos/enzimologia , Interferência de RNA , TransfecçãoRESUMO
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.
Assuntos
Proteínas de Drosophila/genética , Neprilisina/genética , Adulto , Sequência de Aminoácidos/genética , Animais , Linhagem Celular , Bases de Dados de Proteínas , Proteínas de Drosophila/química , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Masculino , Túbulos de Malpighi/química , Túbulos de Malpighi/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Neprilisina/química , Neprilisina/fisiologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Sinais Direcionadores de Proteínas/genética , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Testículo/enzimologia , Testículo/metabolismoRESUMO
We have characterized a phosphatase activity present on the external surface of intact Malpighian tubules in Rhodnius prolixus. This phosphatase hydrolyses the substrate p-nitrophenyl phosphate at a rate of 3.38 +/- 0.07 nmol Pi x mg(-1) x min(-1). Phosphatase activity decreased with the increase of the pH from 6.4 to 7.6 pH, a range in which tubules cellular integrity was maintained for at least 1 h. Classical inhibitors of acid phosphatase, such as ammonium molybdate, fluoride, vanadate, mpV-PIC, and bpV-PHEN, caused different patters of inhibition. The ecto-phosphatase present an apparent Km of 1.67 +/- 0.34 mM and Vmax of 5.71 +/- 0.37 nmol Pi x mg(-1) x min(-1) for p-NPP. Zinc chloride inhibited 78.2% of ecto-phosphatase activity, with Ki of 0.35 mM. Such inhibition was reversed by incubation with cysteine and GSH, but not DTT, serine, and GSSG, showing that cysteine residues are important for enzymatic activity. Phosphatase activity increased 141% three days after blood meal, and returned to basal levels 2 days later. These results suggest that ecto-phosphatase activity could be involved in a diuretic mechanism, essential in the initial days after a blood meal for the control of Rhodnius homeostasis.
Assuntos
Membrana Celular/enzimologia , Túbulos de Malpighi/enzimologia , Estado Nutricional/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Rhodnius/enzimologia , Animais , Concentração de Íons de Hidrogênio , Masculino , Túbulos de Malpighi/citologia , Fatores de TempoRESUMO
cGMP-dependent protein kinase (cGK) forms encoded by the dg2 (for) gene are implicated in behavior and epithelial transport in Drosophila melanogaster. Here, we provide the first biochemical characterization and cellular localization of cGKs encoded by the major transcripts of dg2: dg2P1 and dg2P2. cGMP stimulates kinase activity of DG2P1 (EC(50): 0.13 +/- 0.039 microm) and DG2P2 (EC(50): 0.32 +/- 0.14 microm) in Malpighian tubule and S2 cell extracts. DG2P1 and DG2P2 are magnesium-requiring enzymes and were inhibited by 10 and 100 microm of a cGK inhibitor, 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, Rp isomer; whereas DG1, the cGK encoded by the D. melanogaster dg1 gene, was unaffected. DG2P1 and DG2P2 were localized in the plasma membrane in S2 cells, whereas DG1 was localized in the cytosol. The D. melanogaster fluid-transporting Malpighian tubule was used as an organotypic model to analyze cGK localization and function in vivo. Targeted expression of DG2P2, DG2P1, and DG1 in tubule cells via the UAS/GAL4 system in transgenic flies revealed differential localization of all three cGKs in vivo: DG2P2 expression at the apical membrane; DG2P1 expression at both the apical and basolateral membranes; and DG1 expression at the basolateral membrane and in the cytosol. Transgenic tubules for all three cGKs displayed enhanced cGK activity compared with wild-type tubules. The physiological impact of targeted expression of individual cGKs in tubule principal cells was assessed by measuring basal and stimulated rates of fluid transport. DG1 expression greatly enhanced fluid transport by the tubule in response to exogenous cGMP, whereas DG2P2 expression significantly increased fluid transport in response to the nitridergic neuropeptide, capa-1. Thus, dg2-encoded proteins are bona fide cGKs, which have differential roles in epithelial fluid transport, as assessed by in vivo studies. Furthermore, a novel epithelial role is suggested for DG1, which is considerably more responsive to cGMP than to capa-1 stimulation.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila melanogaster , Células Epiteliais/enzimologia , Rim/metabolismo , Túbulos de Malpighi/enzimologia , Técnicas de Cultura de Órgãos , FenótipoRESUMO
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.
Assuntos
Proteínas de Drosophila/metabolismo , Células Epiteliais/metabolismo , Óxido Nítrico/fisiologia , Diester Fosfórico Hidrolases/metabolismo , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Drosophila melanogaster , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Feminino , Masculino , Túbulos de Malpighi/efeitos dos fármacos , Túbulos de Malpighi/enzimologia , Túbulos de Malpighi/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase/fisiologia , Inibidores de Fosfodiesterase/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
In a previous paper, we observed that the specific activity of (Na++K+)ATPase of the isolated Malpighian tubules from Rhodnius prolixus is inhibited by protein kinase C (PKC) during hyperosmotic shock [Arenstein et al., J Membr Biol 146:47-57 [1995]; Caruso-Neves et al., Z Naturforsch 53c:911-917 [1998]). In the present paper, we study the involvement of the cytoskeleton in this process using isolated Malpighian tubules of Rhodnius prolixus. We observed that pre-incubation of the Malpighian tubule cells in hyperosmotic media decreases the specific activity of (Na++K+)ATPase by 90%. This effect was completely reversed when colchicine, which disrupts microtubules, or cytochalasin B, an inhibitor of actin microfilament polymerization, were added to the media in a dose-dependent manner. The maximal reversion was obtained with colchicine 7.0 microM or cytochalasin B 5.0 microM. The simultaneous addition of sphingosine 50 ng/mL, an inhibitor of PKC, to 10 microM colchicine or 5 microM cytochalasin B, in hyperosmotic media, did not change the stimulatory effect of these drugs on the specific activity of (Na++K+)ATPase. On the other hand, the co-incubation of TPA 20 ng/mL, an activator of PKC, to colchicine or cytochalasin B within hyperosmotic media, abolished the stimulatory effect of these drugs on the specific activity of (Na++K+)ATPase to a similar extent as hyperosmotic shock. These results suggest that inhibition of the (Na++K+)ATPase of the isolated Malpighian tubules from Rhodnius prolixus by PKC during hyperosmotic shock is mediated by cytoskeletal elements.
Assuntos
Túbulos de Malpighi/enzimologia , Proteína Quinase C/metabolismo , Reduviidae/enzimologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Colchicina/farmacologia , Citocalasina B/farmacologia , Citoesqueleto/enzimologia , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Cinética , Masculino , Túbulos de Malpighi/efeitos dos fármacos , Pressão Osmótica , Proteína Quinase C/antagonistas & inibidores , Reduviidae/efeitos dos fármacos , Esfingosina/farmacologiaRESUMO
The Ussing model has long provided the conceptual foundation for understanding epithelial transport mechanisms energized by the Na(+)-K(+)-ATPase. Plasma membranes may also use the vacuolar (V-type) H(+)-ATPase as the primary energy source of membrane and epithelial transport. A pure electrogenic pump, the V-type H(+)-ATPase energizes not only membranes it inhabits but also other transport pathways via electrical coupling.
Assuntos
Túbulos de Malpighi/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Vacúolos/enzimologia , Animais , Transporte Biológico/fisiologia , Epitélio/enzimologiaRESUMO
The Drosophila melanogaster homologue of an insect calcitonin-like diuretic hormone was identified in a BLAST search of the Drosophila genome database. The predicted 31-residue amidated peptide (D. melanogaster DH31; Drome-DH31) was synthesised and tested for activity on fruit fly Malpighian tubules. It increases tubule secretion by approximately 35 % of the response obtained with a myokinin from the housefly Musca domestica (muscakinin; Musdo-K) and has an EC50 of 4.3 nmol x l(-1). The diuretic activities of Drome-DH31 and Musdo-K were additive when tested at threshold and supra-maximal concentrations, which suggests that they target different transport processes. In support of this, Drome-DH31 increased the rate of secretion by tubules held in bathing fluid with a reduced Cl- concentration, whereas Musdo-K did so only in the presence of Drome-DH31. Stimulation with Drome-DH31 increased the lumen-positive transepithelial potential in the main secretory segment of the tubule. This was attributed to activation of an apical electrogenic proton-translocating V-ATPase in principal cells, since it was associated with hyperpolarisation of the apical membrane potential and acidification of secreted urine by 0.25 pH units. Exogenous 8-bromo-cyclic AMP and cyclic GMP increased tubule secretion to the same extent as Drome-DH31 and, when tested together with the diuretic peptide, their activities were not additive. Stimulation with Drome-DH31 resulted in a dose-dependent increase in cyclic AMP production by tubules incubated in saline containing 0.5 mmol x l(-1) 3-isobutyl-1-methylxanthine, whereas cyclic GMP production was unchanged. Taken together, the data are consistent with Drome-DH31 activating an apical membrane V-ATPase via cyclic AMP. Since the K+ concentration of the secreted urine was unchanged, it is likely that Drome-DH31 has an equal effect on K+ and Na+ entry across the basolateral membrane.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/enzimologia , Hormônios de Inseto/farmacologia , Proteínas de Insetos/farmacologia , Túbulos de Malpighi/efeitos dos fármacos , ATPases Translocadoras de Prótons/metabolismo , ATPases Vacuolares Próton-Translocadoras , 1-Metil-3-Isobutilxantina/farmacologia , Sequência de Aminoácidos , Animais , Bucladesina/farmacologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Relação Dose-Resposta a Droga , Feminino , Concentração de Íons de Hidrogênio , Hormônios de Inseto/química , Hormônios de Inseto/genética , Hormônios de Inseto/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Masculino , Túbulos de Malpighi/enzimologia , Túbulos de Malpighi/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Muscidae , Neuropeptídeos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Alinhamento de Sequência , Urina/químicaRESUMO
Malpighian tubule of Rhodnius sp. express two sodium pumps: the classical ouabain-sensitive (Na+ + K+)ATPase and an ouabain-insensitive, furosemide-sensitive Na+-ATPase. In insects, 5-hydroxitryptamine is a diuretic hormone released during meals. It inhibits the (Na+ + K+)ATPase and Na+ -ATPase activities indicating that these enzymes are involved in fluid secretion. Furthermore, in Rhodnius neglectus, proximal cells of Malpighian tubule exposed to hyperosmotic medium, regulate their volume through a mechanism called regulatory volume increase. This regulatory response involves inhibition of the (Na+ + K+)ATPase activity that could lead to accumulation of active osmotic solute inside the cell, influx of water and return to the normal cell volume. Adenosine, a compound produced in stress conditions, also inhibits the (Na+ + K+)ATPase activity. Taken together these data indicate that (Na+ + K+)ATPase is a target of the regulatory mechanisms of water and ions transport responsible for homeostasis in Rhodnius sp.
Assuntos
Túbulos de Malpighi/enzimologia , Rhodnius/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/fisiologiaRESUMO
Malpighian tubule of Rhodnius sp. express two sodium pumps: the classical ouabain-sensitive (Na+ + K+)ATPase and an ouabain-insensitive, furosemide-sensitive Na+-ATPase. In insects, 5-hydroxitryptamine is a diuretic hormone released during meals. It inhibits the (Na+ + K+)ATPase and Na+ -ATPase activities indicating that these enzymes are involved in fluid secretion. Furthermore, in Rhodnius neglectus, proximal cells of Malpighian tubule exposed to hyperosmotic medium, regulate their volume through a mechanism called regulatory volume increase. This regulatory response involves inhibition of the (Na+ + K+)ATPase activity that could lead to accumulation of active osmotic solute inside the cell, influx of water and return to the normal cell volume. Adenosine, a compound produced in stress conditions, also inhibits the (Na+ + K+)ATPase activity. Taken together these data indicate that (Na+ + K+)ATPase is a target of the regulatory mechanisms of water and ions transport responsible for homeostasis in Rhodnius sp.