Lipophorin interaction with the midgut of Rhodnius prolixus: characterization and changes in binding capacity

Several classes of lipids are transported in insect hemolymph by lipophorin, a major hemolymphatic lipoprotein. The binding of lipophorin to the midgut of the hematophagous insect Rhodnius prolixus was characterized in a midgut membrane preparation, using purified lipophorin radiolabelled in protein moiety ((125)I-HDLp). Lipophorin specific binding to membranes achieved equilibrium after 30-40 min, was sensitive to pH, and was maximal at pH 7.0. In the presence of increasing concentrations of membrane protein, corresponding increases in lipophorin binding were observed. The specific binding of lipophorin to the membrane preparation was a saturable process, with K(d)=0.9+/-0.06 x 10(-7) M and a maximal binding capacity of 70+/-11 ng lipophorin/microg of membrane protein. Lipophorin binding did not depend on calcium, but it was affected by ionic strength and was inhibited in the presence of increasing salt concentrations. Suramin interfered with lipophorin binding to the midgut receptor, and it was abolished in the presence of 2 mM suramin, but at concentrations between 0.05 and 0.2 mM it was slightly increased. Condroitin 4-sulfate also affected lipophorin binding, which was reduced to 56% of control. Pre-incubation of the midgut membrane preparation with trypsin or at high temperature inhibited binding. Midgut capacity to bind lipophorin varied at different days after blood meal. It was highest at second day after feeding, and then gradually decreased.     

Na,K-ATPase and V-ATPase in ovarian follicles of Drosophila melanogaster.

Uncovering the cause and meaning of bioelectric phenomena in developing systems requires investigations of the distribution and activity of ion-transport mechanisms. In order to identify and localize ion pumps in ovarian follicles of Drosophila, we used immunofluorescence microscopy, immunoelectron microscopy, subcellular fractionation, immunoblots, and acridine-orange staining. We applied various antibodies directed against the Na,K-pump (Na,K-ATPase) and against vacuolar-type proton pumps (V-ATPase). During all phases of oogenesis, Na,K-ATPase were found in apical and lateral follicle-cell membranes and, during rapid follicle growth (beginning with stage 10), also in nurse-cell membranes and in the oolemma. V-ATPase were detected in various cytoplasmic vesicles and in yolk spheres and, beginning with stage 10, also in apical follicle-cell membranes and in the oolemma. Given these and earlier results, we propose that: 1) V-ATPase coupled to secondary active antiporters represent the ouabain-intensitive potassium pumps described previously; 2) both Na,K-ATPase and V-ATPase are involved in bioelectric phenomena as well as in osmoregulation and follicle growth, especially during stages 10-12; 3) organelle-associated V-ATPase play a role in vesicle acidification and in yolk processing; and 4) the channel-forming protein ductin is a component of both V-ATPase and gap junctions in ovarian follicles of Drosophila.     

The distribution and activity of tachykinin-related peptides in the blood-feeding bug, Rhodnius prolixus

The invertebrate tachykinin-related peptides (TRPs) with the conserved C-terminal sequence FX1GX2Ramide shows sequence similarity to the vertebrate tachykinins after which they are named, and are hypothesized to be ancestrally related. In this study a polyclonal antiserum generated against a locust tachykinin (LomTK I), was used to demonstrate the presence and describe the distribution of LomTK-like immnoreactivity in the CNS and gut of Rhodnius prolixus. Reverse phase high performance liquid chromatography (RP-HPLC) was used in combination with a sensitive radioimmunoassay (RIA) to demonstrate picomolar amounts of immunoreactive material in the CNS, and femptomolar amounts associated with the hindgut. Furthermore, the results from CNS extracts separated by RP-HPLC, suggest that at least two tachykinin isoforms exist in R. prolixus. A hindgut contraction assay was developed to quantify the myotropic effects of selected LomTKs on R. prolixus hindgut contraction. Both LomTK I and II caused an increase in the frequency of hindgut contractions with EC50 values of 3.6x10(-8)M and 3.8x10(-8)M for LomTK I and II, respectively.     

Characterization of gill Na/K-ATPase activity and ouabain binding in Antarctic and New Zealand nototheniid fishes

The effects of thermal acclimation in two Nototheniid species, the stenothermal Antarctic Trematomous bernacchii and the eurythermal New Zealand Notothenia angustata, were investigated. Serum osmolality, gill Na/K-ATPase activity, sodium pump density and ouabain affinity were determined. Both fish were acclimated at their upper and lower viable thermal temperatures. Warm acclimation (+4 degrees C) of the T. bernacchii significantly decreased their serum osmolality from 550 to 450 mOsm/kg compared to cold-acclimation (-1.5 degrees C) and this was accompanied by a two-fold increase in gill Na/K-ATPase activity. Warm-acclimation (+14 degrees C) of N. angustata did not significantly change their serum osmolality from 330 mOsm/kg or gill Na/K-ATPase activity compared to the cold-acclimated (+4 degrees C) N. angustata. Using [(3)H]ouabain binding techniques, the B(max) and K(d) values of gill Na/K-ATPase enzymes were determined. No difference in the B(max) or K(d) of the warm-acclimated T. bernacchii accounted for the increase in Na/K-ATPase activity. We conclude that the change in gill Na/K-ATPase activity in the warm-acclimated T. bernacchii is not mediated by an increase in the number of enzyme sites and is not reflected in a change in ouabain affinity for Na/K-ATPase.     

In vivo modification of Na,K-ATPase activity in Drosophila

We have constructed and characterized transgenic Drosophila lines with modified Na⁺,K⁺-ATPase activity. Using a temperature dependent promoter from the hsp70 gene to drive expression of wild-type α subunit cDNA, we can conditionally rescue bang-sensitive paralysis and ouabain sensitivity of a Drosophila Na⁺,K⁺-ATPase α subunit hypomorphic mutant, 2206. In contrast, a mutant α subunit (α^D369N) leads to increased bang-sensitive paralysis and ouabain sensitivity. We can also generate temperature dependent phenotypes in wild-type Drosophila using the same hsp70 controlled α transgenes. Ouabain sensitivity was as expected, however, both bang sensitive paralysis or locomotor phenotypes became more severe regardless of the type of α subunit transgene. Using the Gal4-UAS system we have limited expression of α transgenes to cell types that normally express a particular Drosophila Na⁺,K⁺-ATPase β (Nervana) subunit isoform (Nrv1 or 2). The Nrv1-Gal4 driver results in lethality while the Nrv2-Gal4 driver shows reduced viability, locomotor function and uncontrolled wing beating. These transgenic lines will be useful for disrupting function in a broad range of cell types.     

Effects of lignoids on a hematophagous bug, Rhodnius prolixus: feeding, ecdysis and diuresis

The effects of lignoids on feeding, ecdysis and diuresis in fourth-instar larvae of Rhodnius prolixus (Hemiptera) were investigated. Up to 100 microg/ml burchellin, podophyllotoxin, pinoresinol, sesamin, licarin A, or nordihydroguaiaretic acid (NDGA) in the diet did not induce antifeedant effects. Pinoresinol and NDGA significantly inhibited ecdysis. In experiments in vivo, burchellin and podophyllotoxin reduced the production of urine after feeding. 5-Hydroxytryptamine (5-HT), a diuretic hormone, partially counteracted this effect of burchellin. In experiments in vitro, using isolated Malpighian tubules, (i) burchellin reduced diuretic hormone levels in the hemolymph but not the amount of diuretic hormone stored in the thoracic ganglionic masses (including axons), (ii) burchellin decreased the volume of urine secreted by isolated Malpighian tubules, and (iii) 5-HT could not overcome the effect of burchellin upon the Malpighian tubules. We conclude that burchellin interfered with the release, but not with the production of diuretic hormone by the thoracic ganglionic mass or induced an antidiuretic hormone and directly affected the Malpighian tubules.     

Characterization and immunocytochemical localization of lipophorin binding sites in the oocytes of Rhodnius prolixus

Purified lipophorin, metabolically labelled with 32P exclusively in the phospholipid moiety, was used to study the process of phospholipid delivery to the oocyte. The kinetics of phospholipid transfer "in vitro," from lipophorin to the oocytes, was linear at least up to 4 h and was impaired by low temperature. A net transfer of phospholipids from lipophorin particles to the oocytes was observed. The rate of phospholipid uptake was dependent on the concentration of lipophorin in the medium and was shown to be a saturable process. The addition of a molar excess of purified unlabelled lipophorin to the culture medium resulted in a substantial decrease in the transfer of [32P]phospholipids, but no reduction occurred in the presence of a molar excess of albumin. The lipophorin binding sites were localized in the oocytes by immunogold techniques using two different protocols for oocyte fixation. Strong labelling was observed especially at the microvilli. No labelling was detected in the yolk granules.     

Effect of various organic substances on the activity of the membrane preparations of Na, K-ATPase]

The effect of guanidine, lisine, arginine, acetamide and urea on the activity of the preparations of Na, K-ATPase from guinea pig kidney was studied. It was established that the enzymatic activity of the preparations can be lowered by 50% by the following concentrations of the substances examined: guanidine--0.07 M, argine--0.12 M, lisine--0.30 M, acetamide--0.95 M, urea--1.05 M. There correlation among the inhibitory ability of these substances and their basis properties.     

Na,K-ATPase function and cellular proliferative activity in culture

A study was made of the dependence of ATP hydrolysis intensity upon different ratios of sodium and potassium ions in plasma membrane of L cells and of cells of clone Lebr 625, sensitive and resistant to ethidium bromide, and of the distribution of cells according to cell cycle phases in dense and sparse cultures. In dense cultures, the cell growth is arrested on G1 phase, the hydrolytic activity of (Na+ + K+)-ATPase decreases, and the Na+, K+ ratio for maximum activity of (Na+ + K+)-ATPase changes. The higher proliferative activity of Lebr 625 cells in dense culture corresponds to the higher hydrolytic activity of (Na+ + K+)-ATPase.     

Presence and activity of a Dippu-DH31-like peptide in the blood-feeding bug, Rhodnius prolixus

The blood-feeding bug, Rhodnius prolixus, ingests large blood meals, then undergoes a period of rapid diuresis which is under neurohormonal control. In both cockroach (Diploptera punctata) and fruit fly (Drosophila melanogaster) a calcitonin-like DH31 neuropeptide has been identified [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795-804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469-74] and demonstrated to be active on Malpighian tubule secretion [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795-804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469-74]. Using an antibody raised against D. punctata (Dippu) DH31, we demonstrate the presence of Dippu-DH31-like immunoreactivity in the CNS, salivary glands, hindgut and neurohemal sites of 5th instar Rhodnius. Double-label immunohistochemistry for Dippu-DH31-like and serotonin-like immunoreactivity demonstrates some co-localization of these factors in cells of the mesothoracic ganglionic mass (MTGM) and in neurohemal sites on the abdominal nerves. When tested on Rhodnius 5th instar Malpighian tubules, Dippu-DH31 stimulated minor increases in rate of secretion. Dippu-DH31 tested in combination with serotonin resulted in increases in the rate of secretion which were at least additive.     

Effects of air ions on the membrane Na, K-ATPase activity of L 1210 cells

The effect of exposure of L 1210 mouse leukemia cells to artificially generated air ions on the activity of membrane Na, K-ATPase in the cells was investigated. The exposure of cells to air ions of both signs gave identical results, i.e. diminution of transport activity of the enzyme, measured by radioactive 86Rb transport into the cell (ouabain-dependent). Passive ouabain-independent transport of Rb+ into the cells remained unchanged in the air ion-treated cells, as did the passive efflux of the radioisotope from preloaded cells. The possible explanation of the phenomena observed is discussed.     

Temporal modulation and adaptive control of the behavioural response to odours in Rhodnius prolixus

It has been demonstrated in several insect species that a circadian clock makes the whole of antennal chemoreceptors more sensitive during a particular temporal window every day. This assessment raises the question about how insects exhibiting bimodal activity handle their sensitivity to odours which are relevant at different moments of the day. To shed some light on this problem, we studied in Rhodnius prolixus the daily dynamics of their responsiveness to CO₂ (host-associated cue) and aggregation cues (refuge-associated), which are relevant at dusk and dawn, respectively. We analysed: (1) whether a temporal modulation of the responsiveness to odours does exist in R. prolixus, (2) if this modulation is a general one or it is specific for each type of volatile, and (3) if it is controlled by exogenous or endogenous mechanisms. We found that the responsiveness to CO₂ only occurs at dusk and that to assembling odours is restricted to dawn. Experiments under free-running conditions revealed that only the responsiveness to CO₂ is controlled by a circadian clock, but not that to assembling signals. Thus, by combining endogenous and exogenous mechanisms, sensitivities to different odours are adjusted according to their associated behavioural context and moment of the day.     

Isoforms of Na,K-ATPase inArtemia saline: I. Detection by FITC binding and time course

Summary Partially purified Na,K-ATPase from whole nauplii at various stages of development, analyzed by SDS-PAGE, reveals a polydisperse and two subunits (denoted 1 and 2). In the absence of Ca²⁺, ATP-inhibitable fluorescein isothiocyanate (FITC) labeling is restricted to the subunit of this enzyme, even in crude naupliar homogenates. The intensity of the -specific fluorescent signal (i.e., the sum of the yield from both isoforms) is proportional to Na,K-ATPase activity during development. FITC-labeled subunits were detected at 8 hr of development prior to the detection of measurable Na,K-ATPase activity. The 2/1 ratio changed from an initial value of 1.25 to a peak of 1.75 at 32 hr of development, then reverted to a ratio of 1.25 by 42 hr, and remained constant thereafter. Pulse chase studies with³⁵S-methionine indicated that the developmental increase in enzyme activity is coincident with amino acid incorporation into the subunits, implying that enzyme synthesis is active during enzyme accumulation.During the tenure of an Educational Commission for Foreign Medical Graduates Visiting Associate Professorship.     

Modulation of Na,K-ATPase and Na-ATPase activity by phospholipids and cholesterol. I. Steady-state kinetics

The effects of phospholipid acyl chain length (n(c)), degree of acyl chain saturation, and cholesterol on Na,K-ATPase reconstituted into liposomes of defined lipid composition are described. The optimal acyl chain length of monounsaturated phosphatidylcholine in the absence of cholesterol was found to be 22 but decreased to 18 in the presence of 40 mol % cholesterol. This indicates that the hydrophobic matching of the lipid bilayer and the transmembrane hydrophobic core of the membrane protein is a crucial parameter in supporting optimal Na,K-ATPase activity. In addition, the increased bilayer order induced by both cholesterol and saturated phospholipids could be important for the conformational mobility of the Na,K-ATPase changing the distribution of conformations. Lipid fluidity was important for several parameters of reconstitution, e.g., the amount of protein inserted and the orientation in the liposomes. The temperature dependence of the Na,K-ATPase as well of the Na-ATPase reactions depends both on phospholipid acyl chain length and on cholesterol. Cholesterol increased significantly both the enthalpy of activation and entropy of activation for Na,K-ATPase activity and Na-ATPase activity of Na,K-ATPase reconstituted with monounsaturated phospholipids. In the presence of cholesterol the free energy of activation was minimum at a lipid acyl chain length of 18, the same that supported maximum turnover. In the case of ATPase reconstituted without cholesterol, the minimum free energy of activation and the maximum turnover both shifted to longer acyl chain lengths of about 22.     

Feeding in Rhodnius prolixus: mouthpart activity and salivation, and their correlation with chanees of electrical resistance

Detailed observations were made of mouth part movements, salivation and ingestion in Rhodnius prolixus feeding on an artificial diet. Several events not previously described in blood-feeding Hemiptera were recorded; these included formation of a salivary ‘collar’, continuous salivation during probing, and sampling of diet prior to initiation of gorging. The events during feeding were analysed in detail and correlated with changes in electrical resistance between haemolymph and diet by recording both simultaneously on video-tape using a split-screen television technique.     

Trypanosoma cruzi: effects of infection on cathepsin D activity in the midgut of Rhodnius prolixus

Cathepsin D activity was estimated in midgut homogenates from Rhodnius prolixus, uninfected and experimentally infected with Trypanosoma cruzi, at different times after blood ingestion. No enzyme activity was found in the anterior midgut and rectum. In the posterior midgut, enzyme activity was found both in lumen and wall. In starved uninfected insects, in lumen and wall, cathepsin D activity was high, decreasing to a constant rate at 1-15 days after feeding. In insects infected with T. cruzi cathepsin D activity increased 1 and 3 days after blood meal. We suggest that these changes in cathepsin D activity in R. prolixus posterior midgut are due to the establishment of T. cruzi infection.     

Effects of gamma irradiation on the development of Trypanosoma rangeli in the vector Rhodnius prolixus

Studies on the effects of gamma radiation on the infectivity of Trypanosoma rangeli (strain H14) for the vector Rhodnius prolixus revealed that (i) the LD(50) (lethal dose for 50% of bugs) for uninfected insects was 4147 rads; (ii) irradiated insects with a dose of 1200 rads subsequently infected with the flagellates exhibited a mortality of 45%, while uninfected irradiated insects showed a mortality of 5%, and infected nonirradiated insects exhibited 10% mortality; (iii) flagellates were present in the hemolymph of irradiated insects 7 days postinfection (p.i.), while in nonirradiated insects the parasites appeared in the hemocoel 18 days p.i.; (iv) T. rangeli infection decreased the number of hemocytes significantly and induced the formation of nodules in the hemolymph of both irradiated and nonirradiated insects; and (v) gamma irradiation affected the ultrastructural organization of the epithelial cells of the small intestine, principally the perimicrovillar membranes and microvilli. In this paper, we discuss the significance of the intestinal microenvironment of R. prolixus with regard to its interaction with T. rangeli.     

Investigation of the effect of phospholipase on Na,K-ATPase activity]

Temperature dependence of bovine brain NA,K-ATPase before and after the short-term treatment of enzyme preparations with phospholipases A, C and D is investigated. Arrhenius plots of the temperature dependence of the reaction rate catalysed by Na,K-ATPase are non-linear, they have an inflection at the region of about 20 degrees C. The treatment of the enzyme with phospholipase A makes the inflection more smooth, phospholipase D shifts the inflection by 4 degrees C to lower temperature and simultaneously activates Na,K-ATPase. Phospholipase C sharply changes the Arrhenius curve and makes it linear. The data obtained are discussed with respect to the role of phospholipids in the formation of membrane bilayer and in the regulation of Na,K-ATPase activity.     

Functional activity of tissue Na,K-ATPase in health and in pathology]

The review deals with two basic possibilities of regulation of ion-transporting activity. The first possibility is connected with the changes in the number of working molecules of Na, K-ATP-ase and the second one with the change in the number of turns of active molecules of the pump.