Effects of leucokinin-VIII on Aedes Malpighian tubule segments lacking stellate cells.
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The diuretic peptide leucokinin is known to increase fluid secretion in Malpighian tubules of the yellow fever mosquito Aedes aegypti by increasing a transepithelial Cl(-) conductance. The present study sought to examine whether stellate cells provided this transepithelial conductance in Aedes Malpighian tubules as they do in Drosophila Malpighian tubules. Aedes Malpighian tubule segments with and without stellate cells were perfused in vitro for measurements of the transepithelial voltage (V(t)), resistance (R(t)) and Cl(-) diffusion potentials (DP(Cl)). In 11 tubule segments containing both principal cells and stellate cells, 1 micro mol l(-1) leucokinin-VIII added to the peritubular bath immediately and significantly decreased V(t) from 39.3+/-14.3 mV to 2.3+/-0.7 mV, decreased R(t) from 12.4+/-2.6 kOmegacm to 2.4+/-0.3 kOmegacm, and increased DP(Cl) from 8.2+/-1.2 mV to 42.1+/-5.4 mV. These effects of leucokinin-VIII were qualitatively and quantitatively similar in six tubule segments containing no stellate cells; V(t) decreased from 37.8+/-7.0 mV to 3.4+/-0.6 mV, R(t) decreased from 8.8+/-2.1 kOmegacm to 1.7+/-0.2 kOmegacm, and DP(Cl) increased from 5.8+/-2.6 mV to 50.0+/-2.1 mV. Thus, stellate cells are not required for signaling or mediating the effects of leucokinin in Malpighian tubules of Aedes aegypti. The results further support previous observations that principal cells signal the effects of leucokinin to increase the Cl(-) conductance of the paracellular pathway through septate (or tight) junctions. (+info)
Inorganic and organic anion transport by insect renal epithelia.
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Insect renal organs typically exhibit high rates of transport of inorganic and organic anions, and therefore provide useful models for the study of epithelial anion transport and its control. Isolated Malpighian tubules of some species secrete a volume of iso-osmotic fluid equal to their own volume in 10-15 s, which means that cellular Cl(-) content is exchanged every 3-5 s. Anion transport can also be achieved against extreme thermodynamic gradients. The concentration of K(+) and Cl(-) in the lumen of the Malpighian tubules of some desert beetles approaches or exceeds saturation. A basolateral Na(+):K(+):2Cl(-) cotransporter plays an important role in vectorial ion transport in Malpighian tubules of many species, but there is also evidence for coupling of Cl(-) transport to the movement of a single cationic species (Na(+) or K(+)). Although an apical vacuolar H(+)-ATPase plays a primary role in energizing transepithelial secretion of chloride via channels or cotransporters in the secretory segment of the Malpighian tubule, several different ATPases have been implicated in reabsorption of Cl(-) by the lower Malpighian tubule or hindgut. Chloride transport is known to be controlled by several neuropeptides, amines and intracellular second messengers. Insect renal epithelia are also important in excretion of potentially toxic organic anions, and the transporters involved may play a role in resistance to insecticides of natural or anthropogenic origin. (+info)
In vivo binding of the Cry11Bb toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae).
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Bacillus thuringiensis subsp. medellin produces numerous proteins among which 94 kDa known as Cry11Bb, has mosquitocidal activity. The mode of action of the Cry11 proteins has been described as similar to those of the Cry1 toxins, nevertheless, the mechanism of action is still not clear. In this study we investigated the in vivo binding of the Cry11Bb toxin to the midgut of the insect species Anopheles albimanus, Aedes aegypti, and Culex quinquefasciatus by immunohistochemical analysis. Spodoptera frugiperda was included as negative control. The Cry11Bb protein was detected on the apical microvilli of the midgut epithelial cells, mostly on the posterior midgut and gastric caeca of the three mosquito species. Additionally, the toxin was detected in the Malpighian tubules of An. albimanus, Ae. aegypti, Cx. quinquefasciatus, and in the basal membrane of the epithelial cells of Ae. aegypti midgut. No toxin accumulation was observed in the peritrophic membrane of any of the mosquito species studied. These results confirm that the primary site of action of the Cry11 toxins is the apical membrane of the midgut epithelial cells of mosquito larvae. (+info)
Mechanisms of K+ transport across basolateral membranes of principal cells in Malpighian tubules of the yellow fever mosquito, Aedes aegypti.
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The mechanisms of K(+) entry from the hemolymph into principal cells of Malpighian tubules were investigated in the yellow fever mosquito, Aedes aegypti. The K(+) channel blocker Ba(2+) (5 mmol l(-1)) significantly decreased transepithelial (TEP) fluid secretion (V(s)) from 0.84 nl min(-1) to 0.37 nl min(-1) and decreased the K(+) concentration in secreted fluid from 119.0 mmol l(-1) to 54.3 mmol l(-1) with no change in the Cl(-) concentration. Even though the Na(+) concentration increased significantly from 116.8 mmol l(-1) to 144.6 mmol l(-1), rates of TEP ion secretion significantly decreased for all three ions. In addition, Ba(2+) had the following significant electrophysiological effects: it depolarized the TEP voltage (V(t)) from 19.4 mV to 17.2 mV, increased the TEP resistance (R(t)) from 6.4 kOhmscm to 6.9 kOhmscm, hyperpolarized the basolateral membrane voltage of principal cells (V(bl)) from -75.2 mV to -88.2 mV and increased the cell input resistance from 363.7 kOhms to 516.3 kOhms. These effects of Ba(2+) reflect the block of K(+) channels that, apparently, are also permeable to Na(+). Bumetanide (100 micro mol l(-1)) had no effect on TEP fluid secretion and electrical resistance but significantly decreased TEP K(+) secretion, consistent with the inhibition of electroneutral Na(+)/K(+)/2Cl(-) cotransport. TEP Na(+) secretion significantly increased because other Na(+) entry pathways remained active. Bumetanide plus Ba(2+) completely inhibited TEP electrolyte and fluid secretion, with fast and slow kinetics reflecting the Ba(2+) block of basolateral membrane K(+) channels and the inhibition of Na(+)/K(+)/2Cl(-) cotransport, respectively. The single and combined effects of Ba(2+) and bumetanide suggest that (1) K(+) channels and Na(+)/K(+)/2Cl(-) cotransport are the primary mechanisms for bringing K(+) into cells, (2) K(+) channels mediate a significant Na(+) influx, (3) Na(+) has as many as four entry pathways and (4) the mechanisms of TEP K(+) and Na(+) secretion are coupled such that complete block of TEP K(+) renders the epithelium unable to secrete Na(+). (+info)
Organic cation transport by Malpighian tubules of Drosophila melanogaster: application of two novel electrophysiological methods.
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Transport of the prototypical organic cation tetraethylammonium (TEA) by the Malpighian tubules, ureters and gut of Drosophila melanogaster was studied using two novel electrophysiological techniques. Both techniques exploited the high selectivity of the cation exchanger potassium tetra-p-chlorophenylborate for tetraalkylammonium compounds relative to inorganic cations such as K(+). In the first technique, TEA fluxes were measured using a non-invasive self-referencing TEA-selective microelectrode positioned in the unstirred layer near the surface of each tissue. TEA fluxes from bath to lumen as large as 6 pmol cm(-2) s(-1) were measured across the lower (reabsorptive) segment of the Malpighian tubule and the ureter bathed in saline containing 0.1 mmol l(-1) TEA. Corresponding bath-to-lumen fluxes across the secretory main segment of the Malpighian tubule and the posterior midgut were approximately 1 pmol cm(-2) s(-1). TEA transport by the lower Malpighian tubule was enhanced by hyperpolarization of the basolateral membrane potential and was inhibited by cimetidine, quinidine, vinblastine and verapamil. In the second technique, TEA concentration was measured using a TEA-selective microelectrode positioned in droplets of fluid secreted by Malpighian tubules set up in saline droplets under oil in a Ramsay assay. Results from the Ramsay assay confirmed the dominant role of the lower Malpighian tubule in net transepithelial secretion of TEA and inhibition of TEA transport by cimetidine. Kinetic parameters (J(max) and K(t)) were determined using both approaches. (+info)
Basolateral ion transport mechanisms during fluid secretion by Drosophila Malpighian tubules: Na+ recycling, Na+:K+:2Cl- cotransport and Cl- conductance.
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Mechanisms of ion transport during primary urine formation by the Malpighian tubule of Drosophila melanogaster were analyzed through measurements of fluid secretion rate, transepithelial ion flux, basolateral membrane potential (V(bl)) and intracellular activities of K(+) (a(K)(i)) and Cl(-) (a(Cl)(i)). Calculation of the electrochemical potentials for both ions permitted assessment of the possible contributions of K(+) channels, Na(+):K(+):2Cl(-) cotransport, and K(+):Cl(-) cotransport, to net transepithelial ion secretion across the basolateral membrane. The data show that passive movement of both K(+) and Cl(-) from cell to bath is favoured across the basolateral membrane, indicating that both ions are actively transported into the cell. Contributions of basolateral K(+) channels or K(+):Cl(-) cotransporters to net transepithelial ion secretion can be ruled out. After prior exposure of tubules to ouabain, subsequent addition of bumetanide reduced fluid secretion rate, K(+) flux and Na(+) flux, indicating a role for a Na(+):K(+):2Cl(-) cotransporter in fluid secretion. Addition of the K(+) channel blocker Ba(2+) had no effect on a(K)(i) or a(Cl)(i). Addition of Ba(2+) unmasked a basolateral Cl(-) conductance and the hyperpolarization of V(bl) in response to Ba(2+) was Cl(-)-dependent. A new model for fluid secretion proposes that K(+) and Cl(-) cross the basolateral membrane through a Na(+)-driven Na(+):K(+):2Cl(-) cotransporter and that most of the Na(+) that enters the cells is returned to the bath through the Na(+)/K(+)-ATPase. (+info)
Analysis of Drosophila cGMP-dependent protein kinases and assessment of their in vivo roles by targeted expression in a renal transporting epithelium.
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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. (+info)
The dg2 (for) gene confers a renal phenotype in Drosophila by modulation of cGMP-specific phosphodiesterase.
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Fluid transport in Drosophila melanogaster tubules is regulated by guanosine 3',5'-cyclic monophosphate (cGMP) signalling. Here we compare the functional effects on tubules of different alleles of the dg2 (foraging or for) gene encoding a cGMP-dependent protein kinase (cGK), and show that the fors allele confers an epithelial phenotype. This manifests itself as hypersensitivity of epithelial fluid transport to the nitridergic neuropeptide, capa-1, which acts through nitric oxide and cGMP. However, there was no significant difference in tubule cGK activity between fors and forR adults. Nonetheless, fors tubules contained higher levels of cGMP-specific phosphodiesterase (cG-PDE) activity compared to forR. This increase in cGMP-PDE activity sufficed to decrease cGMP content in fors tubules compared to forR. Challenge of tubules with capa-1 increases cGMP content in both fors and forR tubules, although the increase from resting cGMP levels is greater in fors tubules. Capa-1 stimulation of tubules reveals a potent inhibition of cG-PDE in both lines, although this is greater in fors; and is sufficient to explain the hypersensitive transport phenotype observed. Thus, polymorphisms at the dg2 locus do indeed confer a cGMP-dependent transport phenotype, but this can best be ascribed to an indirect modulation of cG-PDE activity, and thence cGMP homeostasis, rather than a direct effect on cGK levels. (+info)