Mosquito (Aedes aegypti ) aquaporin, present in tracheolar cells, transports water, not glycerol, and forms orthogonal arrays in Xenopus oocyte membranes.
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Previous results showed that mRNA encoding a putative aquaporin (AQP) (GenBank accession number AF218314) is present in the tracheolar cells associated with female Aedes aegypti Malpighian tubules. In this study, immunohistochemistry detected the protein, AeaAQP, also in tracheolar cells, suggesting its involvement in water movement in the respiratory system. When expressed in Xenopus oocytes, AeaAQP increased the osmotic water permeability from 15 x 10(-6) to 150 x 10(-6) m x s-1, which was inhibited by mercury ions. No permeability to glycerol or other solute was observed. AeaAQP expressed in oocytes was solubilized as a homotetramer in nondenaturing detergent as deduced from velocity centrifugation on density gradients. Phylogenetic analysis of MIP (major intrinsic protein) family sequences shows that AeaAQP clusters with other native orthogonal array forming proteins. Specific orthogonal arrays were detected by freeze-fracture analysis of AeaAQP oocyte membranes. We conclude that, in tracheolar cells of A. aegypti, AeaAQP is probably a highly water-permeable homotetrameric MIP which natively can form 2D crystals. (+info)
NorpA and itpr mutants reveal roles for phospholipase C and inositol (1,4,5)- trisphosphate receptor in Drosophila melanogaster renal function.
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Mutants of norpA, encoding phospholipase C beta (PLC beta), and itpr, encoding inositol (1,4,5)-trisphosphate receptor (IP(3)R), both attenuate response to diuretic peptides of Drosophila melanogaster renal (Malpighian) tubules. Intact tubules from norpA mutants severely reduced diuresis stimulated by the principal cell- and stellate cell-specific neuropeptides, CAP(2b) and Drosophila leucokinin (Drosokinin), respectively, suggesting a role for PLC beta in both these cell types. Measurement of IP(3) production in wild-type tubules and in Drosokinin-receptor-transfected S2 cells stimulated with CAP(2b) and Drosokinin, respectively, confirmed that both neuropeptides elevate IP(3) levels. In itpr hypomorphs, basal IP(3) levels are lower, although CAP(2b)-stimulated IP(3) levels are not significantly reduced compared with wild type. However, CAP(2b)-stimulated fluid transport is significantly reduced in itpr alleles. Rescue of the itpr(90B.0) allele with wild-type itpr restores CAP(2b)-stimulated fluid transport levels to wild type. Drosokinin-stimulated fluid transport is also reduced in homozygous and heteroallelic itpr mutants. Measurements of cytosolic calcium levels in intact tubules of wild-type and itpr mutants using targeted expression of the calcium reporter, aequorin, show that mutations in itpr attenuated both CAP(2b)- and Drosokinin-stimulated calcium responses. The reductions in calcium signals are associated with corresponding reductions in fluid transport rates. Thus, we describe a role for norpA and itpr in renal epithelia and show that both CAP(2b) and Drosokinin are PLC beta-dependent, IP(3)-mobilising neuropeptides in Drosophila. IP(3)R contributes to the calcium signalling cascades initiated by these peptides in both principal and stellate cells. (+info)
Effect of sequential heat and cold shocks on nuclear phenotypes of the blood-sucking insect, Panstrongylus megistus (Burmeister) (Hemiptera, Reduviidae).
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Thermal shocks induce changes in the nuclear phenotypes that correspond to survival (heterochromatin decondensation, nuclear fusion) or death (apoptosis, necrosis) responses in the Malpighian tubules of Panstrongylus megistus. Since thermal tolerance increased survival and molting rate in this species following sequential shocks, we investigated whether changes in nuclear phenotypes accompanied the insect survival response to sequential thermal shocks. Fifth instar nymphs were subjected to a single heat (35 or 40 degrees C, 1 h) or cold (5 or 0 degrees C, 1 h) shock and then subjected to a second shock for 12 h at 40 or 0 degrees C, respectively, after 8, 18, 24 and 72 h at 28 degrees C (control temperature). As with specimen survival, sequential heat and cold shocks induced changes in frequency of the mentioned nuclear phenotypes although their patterns differed. The heat shock tolerance involved decrease in apoptosis simultaneous to increase in cell survival responses. Sequential cold shocks did not involve cell/nuclear fusion and even elicited increase in necrosis with advancing time after shocks. The temperatures of 40 and 0 degrees C were more effective than the temperatures of 35 and 5 degrees C in eliciting the heat and cold shock tolerances, respectively, as shown by cytological analysis of the nuclear phenotypes. It is concluded that different sequential thermal shocks can trigger different mechanisms of cellular protection against stress in P. megistus, favoring the insect to adapt to various ecotopes. (+info)
K(+) transport in Malpighian tubules of Tenebrio molitor L: a study of electrochemical gradients and basal K(+) uptake mechanisms.
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Malpighian tubules of the mealworm Tenebrio molitor were isolated for intracellular measurement of basolateral (V(bl)) and, indirectly, apical (V(ap)) membrane potentials. In control Ringer (50 mmol l(-1) K(+), 140 mmol l(-1) Na(+)), V(bl) was 24 mV, cell negative, and V(ap) was 48 mV, cell negative with reference to the lumen. Ion substitution experiments involving K(+) and Na(+) indicated that both V(bl) and V(ap) were sensitive to the bathing K(+) concentration, with the change in V(ap) being 60-77% that of V(bl). A 10-fold drop in bath [K(+)] irreversibly decreased fluid secretion rates from 6.38+/-0.95 nl x min(-1) (mean +/- S.E.M.) to 1.48+/-0.52 nl x min(-1) (N=8). In the presence of 6 mmol l(-1) Ba(2+), a blocker of basal K(+) channels, fluid secretion rates reversibly decreased and the hyperpolarization of both V(bl) and V(ap) seen in 50 mmol l(-1) and 140 mmol l(-1) K(+) indicated a favourable electrochemical gradient for basal K(+) entry. In 5 mmol l(-1) K(+), Ba(2+) induced two different responses: V(bl) either hyperpolarized by approximately 10 mV or depolarised by approximately 14 mV, according to the electrochemical gradient for K(+), which was either inward or outward in low bath [K(+)]. Rubidium, a 'permeant' potassium substitute, caused a hyperpolarization of V(bl), indicating the specificity of K(+) channels found in Tenebrio tubule cells. Other possible K(+) uptake mechanisms located in the basolateral membrane were investigated. Blocking of the putative electroneutral Na(+)/K(+)/2Cl(-) cotransporter by 10 micromol l(-1) bumetanide reversibly decreased fluid secretion rates, with no detectable change in membrane potentials. Ouabain (1 mmol l(-1)), an Na(+)/K(+)-ATPase inhibitor, irreversibly decreased fluid secretion rates but had no effect on electrical potential differences either in the absence or presence of Ba(2+). The results implicate K(+) channels, the Na(+)/K(+)/2Cl(-) contransporter and the Na(+)/K(+)-ATPase in basal K(+) and fluid transport of Tenebrio tubule cells. (+info)
K(+) transport in Malpighian tubules of Tenebrio molitor L: is a K(ATP) channel involved?
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The presence of ATP-regulated K(+) (K(ATP)) channels in Tenebrio molitor Malpighian tubules was investigated by examining the effect of glibenclamide on both fluid secretion and basolateral membrane potentials (V(bl)). Glibenclamide, a K(ATP) channel blocker, slowed fluid secretion of Tenebrio tubules. In low bath K(+) concentration (5 mmol l(-1)), glibenclamide either hyperpolarized or depolarized V(bl), resembling the effect seen with Ba(2+). Subsequent addition of 6 mmol l(-1) Ba(2+) caused a further hyper- or depolarization of V(bl). In control Ringer (50 mmol l(-1) KCl, 90 mmol l(-1) NaCl), glibenclamide had no visible effect on V(bl). The effect of ouabain was investigated in low bath [K(+)] in the presence of Ba(2+). V(bl) responded by a small but significant hyperpolarization from -51+/-4 mV to -56+/-4 mV (n=16, P<0.001) in response to 1 mmol l(-1) ouabain. Repeating the experiments in the presence of both glibenclamide and Ba(2+) resulted in a depolarization of V(bl) when ouabain was added. In low bath [K(+)] (high Na(+)), the Na(+)/K(+)-ATPase is expected to function at a high rate. In the presence of Ba(2+), replacing Na(+) by K(+) rapidly depolarized V(bl), but this was followed by a repolarization. Repeating the experiments in the presence of glibenclamide markedly reduced the depolarizing effect and abolished the repolarization, with a gradual decrease in the sensitivity of V(bl) to the surrounding [K(+)]. These results suggest the presence of K(ATP) channels in the basolateral membrane. Glibenclamide had no visible effect on V(bl) in high K(+) or in the absence of Ba(2+), indicating that other highly conductive K(+) channels may mask the effect on K(ATP) channels. This is the first demonstration of the presence of K(ATP) channels in an insect epithelium. (+info)
Regulation of heat shock proteins, Hsp70 and Hsp64, in heat-shocked Malpighian tubules of Drosophila melanogaster larvae.
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It is known from earlier studies that the heat shock (HS) response in Malpighian tubules (MTs) of Drosophila larvae is different from that in other tissues because instead of the Hsp70 and other common heat shock proteins, Hsp64 and certain other new proteins are induced immediately after HS. In the present study, we examined the kinetics of the synthesis of Hsp70 and Hsp64 immediately after HS and during recovery from HS by 35S-methionine labeling and Western blotting. In addition, we also examined the transcriptional activity of hsp70 genes in larval MT cells at different times after HS by in situ hybridization and Northern blotting. The HS-induced synthesis of Hsp64 ceased by 1 hour of recovery from the HS when synthesis of the Hsp70 commenced. Our results revealed that the induced synthesis of Hsp64 immediately after HS was dependent on new transcription. Although the levels of Hsp70 in MT cells rapidly increased after its synthesis began during recovery, the levels of Hsp64 remained unaltered irrespective of its new synthesis occurring during or after HS. Inhibition of new Hsp64 synthesis by transcriptional or translational inhibitors also did not affect the total amount of this protein in MTs. The Hsp64 polypeptides synthesized in response to HS are degraded rapidly. Apparently, the cells in MTs maintain a balance between new synthesis of Hsp64 and its turnover so that under all conditions a more or less constant level of this protein is maintained. Although the Hsp70 synthesis started only after 1 hour of recovery, the hsp70 genes were transcriptionally activated immediately after HS and they continued to transcribe till at least 4 hours after the HS. The hsp70 transcripts in MT cells that recovered for 2 hours or longer did not contain the 3' untranslated regions (UTRs), which may allow their longer stability and translatability at normal temperature. Synthesis of Hsp70 during recovery period was dependent on continuing transcription. Assessment of the beta-galactosidase activity in 2 transgenic lines carrying the LacZ reporter gene under hsp70 promoter and different lengths of the 5'UTR suggested that the delayed translation of hsp70 transcripts in MTs is probably regulated by some elements in the 5'UTR. (+info)
The action of the excretory apparatus of Calliphora vomitoria in handling injected sugar solution.
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Recent evidence suggests that the isolated Malpighian tubules of Calliphora possess mechanisms which restrict the loss of glucose and trehalose from the insect. This report establishes that the intact, diuresing fly does not excrete glucose or trehalose when solutions of these sugars are injected. When solutions of non-metabolized sugars such as sorbose and xylose are injected into the fly, these sugars are rapidly excreted. High concentrations of sorbose and xylose are found in the urine, suggesting that rapid reabsorption of fluid occurs in the excretory apparatus even during the diuresis which the injections provoke. However, injected sucrose is apparently not excreted in large amounts and it is possible that the Malpighian tubules when functioning in vivo are impermeable to disaccharides. (+info)
The V-type H(+)-ATPase in Malpighian tubules of Aedes aegypti: localization and activity.
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The V-type H(+)-ATPase is thought to provide the driving force for transepithelial electrolyte and fluid secretion in Malpighian tubules. To confirm the presence of this proton pump in Malpighian tubules of the yellow fever mosquito Aedes aegypti, we used several antibodies raised against the V-type H(+)-ATPase of Manduca sexta. Western blot analysis confirmed the presence of the V-type H(+)-ATPase in Malpighian tubules of Aedes aegypti. In situ immunostaining identified the V-type H(+)-ATPase at the apical membrane of the mitochondrion-rich brush border of principal cells. The V-type H(+)-ATPase was not found in stellate cells. Measurements of ATPase activity revealed that bafilomycin-sensitive and NO(3)(-)-sensitive ATPase activity accounted for 50-60% of total ATPase activity in crude extracts of Malpighian tubules. No significant ouabain- or vanadate-sensitive Na(+)/K(+)-ATPase activity was detected. These results support the conclusion reached previously in electrophysiological studies that the mechanisms for transepithelial electrolyte secretion in the Aedes Malpighian tubules rely on the V-type H(+)-ATPase as the principal energizer of epithelial transport. Measures of transepithelial Na(+) and K(+) secretion and estimates of the H(+) flux mediated by the V-type H(+)-ATPase suggest a 1:1 stoichiometry for Na(+)/H(+) and K(+)/H(+) exchange transport across the apical membrane. (+info)