Assessment of the physiological performance of the invasive oriental shrimp Palaemon macrodactylus from an atypical marine population.

Since 2000, a well-established population of the invasive oriental shrimp Palaemon macrodactylus has been present in fully marine conditions in the southwestern Atlantic Ocean (~38° S). To assess the physiological performance of this atypical population restricted to fully marine conditions, we conducted a laboratory experiment in which individuals were transferred from 35 ‰S (local seawater) to 2 ‰S; 5 ‰S; 10 ‰S; 20 ‰S; 50 ‰S and 60‰ for short (6 h), medium (48 h), and long (>504 h) acclimation periods. We measured the time course response of relevant parameters in the shrimp's hemolymph; activity of Na+, K+-ATPase (NKA), and V-H+-ATPase (VHA); and muscle water content. Shrimp showed great osmoregulatory plasticity, being able to survive for long periods between 5 ‰S and 50 ‰S, whereas no individual survived after transfer to either 2 ‰S or 60 ‰S. Shrimp hyper-regulated hemolymph osmolality at 5 ‰S and 10 ‰S, hypo-regulated at 35 ‰S and 50 ‰S, and isosmoticity was close to 20 ‰S. Compared to 35 ‰S, prolonged acclimation to 5 ‰S caused a decrease in hemolymph osmolality (~34%) along with sodium and chloride concentrations (~24%); the NKA and VHA activities decreased by ~52% and ~88%, respectively, while muscle water content was tightly regulated. Our results showed that the atypical population of P. macrodactylus studied here lives in a chronic hypo-osmo-ion regulatory state and suggest that fully marine conditions contribute to its poor performance at the lower limit of salinity tolerance (<5 ‰S).

A kinetic characterization of the gill V(H+)-ATPase from two hololimnetic populations of the Amazon River shrimp Macrobrachium amazonicum.

This investigation examines the kinetic characteristics and effect of acclimation to a brackish medium (21 ‰S) on gill V(H+)-ATPase activity in two hololimnetic populations of M. amazonicum. We also investigate the cellular immunolocalization of the enzyme. Immunofluorescence findings demonstrate that the V(H+)-ATPase c-subunit is distributed in the apical pillar cells of shrimps in fresh water but is absent after acclimation to 21 ‰S for 10 days. V(H+)-ATPase activity from the Tietê River population is ≈50% greater than the Grande River population, comparable to a wild population from the Santa Elisa Reservoir, but is 2-fold less than in cultivated shrimps. V(H+)-ATPase activity in the Tietê and the Grande River shrimps is abolished after 21 ‰S acclimation. The apparent affinities of the V(H+)-ATPase for ATP (0.27 ± 0.04 and 0.16 ± 0.03 mmol L-1, respectively) and Mg2+ (0.28 ± 0.05 and 0.14 ± 0.02 mmol L-1, respectively) are similar in both populations. The absence of V(H+)-ATPase activity in salinity-acclimated shrimps and its apical distribution in shrimps in fresh water underpins the importance of the crustacean V(H+)-ATPase for ion uptake in fresh water.

Rotenoids from Clitoria fairchildiana R. Howard (Fabaceae) seeds affect the cellular metabolism of larvae of Aedes aegypti L. (Culicidae).

Non-domesticated species may represent a treasure chest of defensive molecules which must be investigated and rescued. Clitoria fairchildiana R. Howard is a non-domesticated Fabacea, native from the Amazonian Forest whose seeds are exquisitely refractory to insect predation. Secondary metabolites from these seeds were fractionated by different organic solvents and the CH2Cl2 fraction (CFD - Clitoria fairchildiana dichloromethane fraction), as the most toxic to 3rd instar Aedes aegypti larvae (LC50 180 PPM), was subjected to silica gel chromatography, eluted with a gradient of CH2Cl2: MeOH and sub fractioned in nine fractions (CFD1 - CFD9). All obtained fractions were tested in their toxicity to the insect larvae. Two rotenoids, a 11α-O-ß-D-glucopyranosylrotenoid and a 6-deoxyclitoriacetal 11-O-n-glucopyranoside, were identified in the mixture of CFD 7.4 and CFD 7.5, and they were toxic (LC50 120 PPM) to 3rd instar Ae. aegypti larvae, leading to exoskeleton changes, cuticular detachment and perforations in larval thorax and abdomen. These C. fairchildiana rotenoids interfered with the acidification process of cell vesicles in larvae midgut and caused inhibition of 55% of V-ATPases activity of larvae treated with 80 PPM of the compounds, when compared to control larvae. The rotenoids also led to a significant increase in the production of reactive oxygen species (ROS) in treated larvae, especially in the hindgut region of larvae intestines, indicating a triggering of an oxidative stress process to these insects.

Physicochemical properties of the dissolved organic carbon can lead to different physiological responses of zebrafish (Danio rerio) under neutral and acidic conditions.

Previous studies have suggested that the capacity of natural dissolved organic carbon (DOC) molecules to interact with biological membranes is associated with their aromaticity (SAC340 ); origin (allochthonous versus autochthonous, FI); molecular weight (Abs254/365 ); and relative fluorescence of DOC moieties (PARAFAC analysis). These interactions may be especially important when fish are challenged by acidic waters, which are known to inhibit the active uptake of Na+ and Cl- , while stimulating diffusive ion losses in freshwater fishes. Therefore, zebrafish were acclimated (7 days, pH 7.0) to five natural DOC sources (10 mg C/L), two from the Amazon Basin and three from Canada, together with a "no-added DOC" control. After the acclimation, fish were challenged by exposure to acidic water (pH 4.0) for 3 h. Osmoregulatory parameters were measured at pH 7.0 and 4.0. Acclimation to the five DOC sources did not disturb Na+ , Cl- and ammonia net fluxes, but resulted in differential elevations in Na+ , K+ ATPase and v-type H+ ATPase activities in fish at pH 7.0. However, after transfer to pH.4.0, the control fish exhibited rapid increases in both enzymes. In contrast the DOC- acclimated animals exhibited unchanged (Na+ , K+ ATPase) or differentially increased (v-type H+ ATPase) activities. Na+ , Cl- and ammonia net fluxes remained unchanged in the control fish, but were differentially elevated in most of the DOC treatments at pH 4.0, relative to the same DOC treatments at pH 7.0. Correlations between the osmoregulatory data the DOCs properties highlight that the DOC properties drive different effects on gill physiology.

MPK6 Kinase Regulates Plasma Membrane H+-ATPase Activity in Cold Acclimation.

Cold and freezing stresses severely affect plant growth, development, and survival rate. Some plant species have evolved a process known as cold acclimation, in which plants exposed to temperatures above 0 °C trigger biochemical and physiological changes to survive freezing. During this response, several signaling events are mediated by transducers, such as mitogen activated protein kinase (MAPK) cascades. Plasma membrane H+-ATPase is a key enzyme for the plant cell life under regular and stress conditions. Using wild type and mpk3 and mpk6 knock out mutants in Arabidopsis thaliana, we explored the transcriptional, translational, and 14-3-3 protein regulation of the plasma membrane H+-ATPase activity under the acclimation process. The kinetic analysis revealed a differential profiling of the H+-ATPase activity depending on the presence or absence of MPK3 or MPK6 under non-acclimated or acclimated conditions. Negative regulation of the plasma membrane H+-ATPase activity was found to be exerted by MPK3 in non-acclimated conditions and by MPK6 in acclimated conditions, describing a novel form of regulation of this master ATPase. The MPK6 regulation involved changes in plasma membrane fluidity. Moreover, our results indicated that MPK6 is a critical regulator in the process of cold acclimation that leads to freezing tolerance and further survival.

Sphingolipid Effects on the Plasma Membrane Produced by Addition of Fumonisin B1 to Maize Embryos.

Fumonisin B1 is a mycotoxin produced by Fusarium verticillioides that modifies the membrane properties from animal cells and inhibits complex sphingolipids synthesis through the inhibition of ceramide synthase. The aim of this work was to determine the effect of Fumonisin B1 on the plant plasma membrane when the mycotoxin was added to germinating maize embryos. Fumonisin B1 addition to the embryos diminished plasma membrane fluidity, increased electrolyte leakage, caused a 7-fold increase of sphinganine and a small decrease in glucosylceramide in the plasma membrane, without affecting phytosphingosine levels or fatty acid composition. A 20%-30% inhibition of the plasma membrane H+-ATPase activity was observed when embryos were germinated in the presence of the mycotoxin. Such inhibition was only associated to the decrease in glucosylceramide and the addition of exogenous ceramide to the embryos relieved the inhibition of Fumonisin B1. These results indicate that exposure of the maize embryos for 24 h to Fumonisin B1 allowed the mycotoxin to target ceramide synthase at the endoplasmic reticulum, eliciting an imbalance of endogenous sphingolipids. The latter disrupted membrane properties and inhibited the plasma membrane H+-ATPase activity. Altogether, these results illustrate the mode of action of the pathogen and a plant defense strategy.

Osmoregulatory disturbance in Neotropical fish exposed to the crude extracts of the cyanobacterium, Radiocystis fernandoi.

Blooms of cyanobacteria, a common event in eutrophic environments, result in the release of potentially toxic substances into the water. The cyanobacterium Radiocystis fernandoi produces microcystin (MC) and other peptides that may disturb homeostasis. This study evaluated the effect of intraperitoneal injections containing the crude extract (CE) of R. fernandoi strain R28 on the gills and kidneys of neotropical fish, Piaractus mesopotamicus, 3, 6 and 24 h post-injection. CE contained MC-RR, MC-YR and minor other oligopeptides. Plasma ions and the activities of the enzymes PP1 and PP2A, Na+/K+-ATPase (NKA), H+-ATPase (HA) and carbonic anhydrase (CA) were determined and morphological changes in both the gills and kidneys were characterized. Compared to controls, the concentration of Na+ within the plasma of P. mesopotamicus decreased after treatment with CE 3 h post treatment and increased after 24 h; the concentration of K+ decreased after 6 h. The activity of the endogenous PP1 and PP2A was unchanged in the gills and was inhibited in the kidneys 6 h after i.p. injection. In the gills, NKA activity increased after 3 h and decreased 6 h post i.p. exposure. Further, NKA activity did not differ from the controls 24-h post injection. In the kidneys, NKA, HA and CA activities were unaffected by treatment. The mitochondria-rich cell (MRC) density in the gills decreased after 3 h in the filament and 3 and 6 h in the lamellae and was restored to the control levels 24 h post-exposure. Filament epithelial hyperplasia and hypertrophy, lamellar atrophy and rupture of the lamellar epithelium were the most common effects of treatment in the gills. No histopathological changes occurred in the kidneys. This study demonstrates that a single dose of toxic CE from R. fernandoi can cause a transitory ion imbalance in P. mesopotamicus which is related to the changes in MRC levels and NKA activity. Ionic balance was recovered 24 h post i.p. injection, however, morphological changes that occurred in the gills took a longer amount of time to return to normal. To conclude, the effects of components contained within the CE of R. fernandoi may be harmful to P. mesopotamicus. In particular, the recovery of ionic regulation depends on MRC responses and histopathological changes produced by CE may affect gas exchange and other gill functions.

Dark septate endophytic fungi increase the activity of proton pumps, efficiency of 15N recovery from ammonium sulphate, N content, and micronutrient levels in rice plants.

Plants colonised by dark septate endophytic (DSE) fungi show increased uptake of nutrients available in the environment. The objective of the present study was to evaluate the impact of DSE fungi on the activity of proton pumps, nitrogen (N) recovery from ammonium sulphate, and nutrient accumulation in rice plants. Treatments consisted of non-inoculated plants and plants inoculated with two isolates of DSE fungi, A101 and A103. To determine N recovery from the soil, ammonium sulphate enriched with 15N was added to a non-sterile substrate while parameters associated with the activity of proton pumps and with NO3- uptake were determined in a sterile environment. The A101 and A103 fungal isolates colonised the roots of rice plants, promoting 15N uptake, growth, and accumulation of nutrients as compared with the mock control. A103 induced the expression of the plasma membrane H+-ATPase (PM H+-ATPase) isoforms OsA5 and OsA8, the activity of the PM H+-ATPase and H+-pyrophosphatase. Our results suggest that the inoculation of rice plants with DSE fungi represents a strategy to improve the N recovery from ammonium sulphate and rice plant growth through the induction of OsA5 and OsA8 isoforms and stimulation of the PM H+-ATPase and H+-pyrophosphatase.

The Oligomeric State of the Plasma Membrane H⁺-ATPase from Kluyveromyces lactis.

The plasma membrane H⁺-ATPase was purified from the yeast K. lactis. The oligomeric state of the H⁺-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H⁺-ATPase hexamer in both MASs as the sole/main oligomeric state-in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H⁺-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H⁺-ATPase. It is concluded that the quaternary structure of the H⁺-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer.

Calcium-sensing receptor (CaSR) modulates vacuolar H+-ATPase activity in a cell model of proximal tubule.

BACKGROUND: The calcium-sensing receptor (CaSR) is localized in the apical membrane of proximal tubules in close proximity to the transporters responsible for proton secretion. Therefore, the aim of the present study was to analyze the effects of CaSR stimulation on the biochemical activity of the vacuolar H+-ATPase in a cellular model of proximal tubule cells, OKP cells. METHODS: Biochemical activity of H+-ATPase was performed using cell homogenates, and the inorganic phosphate released was determined by a colorimetric method. Changes in cytosolic ionized calcium [Ca2+]i were also determined using Fluo-4. RESULTS: A significant increase of vacuolar H+-ATPase activity was observed when the CaSR was stimulated with agonists such as Gd3+ (300 µM) and neomycin (200 µM). This activity was also stimulated in a dose-dependent fashion by changes in extracellular Ca2+ (Ca2+o) between 10-4 and 2 mM. Gd3+ and neomycin produced a sustained rise of [Ca2+]i, an effect that disappears when extracellular calcium was removed in the presence of 0.1 µM thapsigargin. Inhibition of phospholipase C (PLC) activity with U73122 (5 × 10-8 M) reduced the increase in [Ca2+]i induced by neomycin. CONCLUSION: CaSR stimulation induces an increase in the vacuolar H+-ATPase activity of OKP cells, an effect that involves an increase in [Ca2+]i and require phospholipase C activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially enhancing its reabsorption in distal tubule segments and reducing the formation of calcium phosphate stones.

Mitochondria-rich cells adjustments and ionic balance in the Neotropical fish Prochilodus lineatus exposed to titanium dioxide nanoparticles.

Manufactured titanium dioxide nanoparticles (TiO2-NP) have been intensely applied in numerous industrial products and may be a risk for aquatic systems as they are not completely removed from domestic and industrial wastes after water treatment. This study evaluated the osmo- and ionic balance, Na+/K+-ATPase, H+-ATPase and carbonic anhydrase activities and the mitochondria-rich cells (MRC) in the gills and kidney of the Neotropical fish Prochilodus lineatus after 2 (acute) and 14 (subchronic) days of exposure to nominal 0, 1, 5, 10 and 50 mg L-1 TiO2-NP. The nominal concentrations corresponded to 0.0, 0.6, 1.6, 2.7 and 18.1 mg L-1 suspended TiO2-NP, respectively, in the water column one hour after NP introduction and were maintained for at least 24 h. Acute exposure to TiO2-NP decreased plasma osmolality and Ca2+ levels. Na+/K+-ATPase, H+-ATPase and carbonic anhydrase activities were inhibited in the gills, but not in the kidney. Total MRC density did not change in gills and kidneys. At gill surface, total MRC density decreased in fish exposed to 50 mg L-1 TiO2-NP and the total MRC fractional surface area unchanged although, there were some changes in the fractional area of MRC with apical microvilli (MRCm) and MRC with apical sponge-like structure (MRCs). MRCm was more abundant than MRCs. After subchronic exposure, there was no change in plasma osmolality, ionic balance and enzyme activities. Total gill MRC density increased in the filament epithelium and renal tubules. In the gills, MRC contacting water exhibited some adjustments. Total MRC and fractional surface area unchanged, but there was an increase of MRCs contacting water at gill surface after exposure to10 and 50 mg L-1 TiO2-NP. MRC proliferation in filament epithelium and in renal tubules as well as the increasing MRCs at gill surface may have contributed to avoid change in plasma osmolality, ionic balance and enzyme activities and suggested a cellular physiological and morphological response to restore and maintain osmotic and ionic homeostasis after subchronic exposure.

High waterborne Mg does not attenuate the toxic effects of Fe, Mn, and Ba on Na+ regulation of Amazonian armored catfish tamoatá (Hoplosternum litoralle).

Formation water (FoW) is a by-product from oil and gas production and usually has high concentrations of soluble salts and metals. Calcium (Ca) and magnesium (Mg) have been shown to reduce the toxicity of metals to aquatic animals, and previous study showed that high waterborne Ca exerts mild effect against disturbances on Na+ regulation in Amazonian armored catfish tamoatá (Hoplosternum littorale) acutely exposed to high Fe, Mn, and Ba levels. Here, we hypothesized that high Mg levels might also reduce the toxic effects of these metals on Na+ regulation of tamoatá. The exposure to 5% FoW promoted an increase in Na+ uptake and a rapid accumulation of Na+ in all tissues analyzed (kidney

Lpx1p links glucose-induced calcium signaling and plasma membrane H+-ATPase activation in Saccharomyces cerevisiae cells.

In yeast, as in other eukaryotes, calcium plays an essential role in signaling transduction to regulate different processes. Many pieces of evidence suggest that glucose-induced activation of plasma membrane H+-ATPase, essential for yeast physiology, is related to calcium signaling. Until now, no protein that could be regulated by calcium in this context has been identified. Lpx1p, a serine-protease that is also involved in the glucose-induced activation of the plasma membrane H+-ATPase, could be a candidate to respond to intracellular calcium signaling involved in this process. In this work, by using different approaches, we obtained many pieces of evidence suggesting that the requirement of calcium signaling for activation of the plasma membrane H+-ATPase is due to its requirement for activation of Lpx1p. According to the current model, activation of Lpx1p would cause hydrolysis of an acetylated tubulin that maintains the plasma membrane H+-ATPase in an inactive state. Therefore, after its activation, Lpx1p would hydrolyze the acetylated tubulin making the plasma membrane H+-ATPase accessible for phosphorylation by at least one protein kinase.

The plasma membrane H+-ATPase gene family in Solanum tuberosum L. Role of PHA1 in tuberization.

This study presents the characterization of the plasma membrane (PM) H+-ATPases in potato, focusing on their role in stolon and tuber development. Seven PM H+-ATPase genes were identified in the Solanum tuberosum genome, designated PHA1-PHA7. PHA genes show distinct expression patterns in different plant tissues and under different stress treatments. Application of PM H+-ATPase inhibitors arrests stolon growth, promotes tuber induction, and reduces tuber size, indicating that PM H+-ATPases are involved in tuberization, acting at different stages of the process. Transgenic potato plants overexpressing PHA1 were generated (PHA1-OE). At early developmental stages, PHA1-OE stolons elongate faster and show longer epidermal cells than wild-type stolons; this accelerated growth is accompanied by higher cell wall invertase activity, lower starch content, and higher expression of the sucrose-H+ symporter gene StSUT1. PHA1-OE stolons display an increased branching phenotype and develop larger tubers. PHA1-OE plants are taller and also present a highly branched phenotype. These results reveal a prominent role for PHA1 in plant growth and development. Regarding tuberization, PHA1 promotes stolon elongation at early stages, and tuber growth later on. PHA1 is involved in the sucrose-starch metabolism in stolons, possibly providing the driving force for sugar transporters to maintain the apoplastic sucrose transport during elongation.

Effects of humic acids from landfill leachate on plants: An integrated approach using chemical, biochemical and cytogenetic analysis.

Biological process treatment of landfill leachate produces a significant amount of sludge, characterized by high levels of organic matter from which humic acids are known to activate several enzymes of energy metabolism, stimulating plant growth. This study aimed to characterize humic acids extracted from landfill sludge and assess the effects on plants exposed to different concentrations (0.5, 1, 2 and 4 mM C L-1) by chemical and biological analysis, to elucidate the influence of such organic material and minimize potential risks of using sludge in natura. Landfill humic acids showed high carbon and nitrogen levels, which may represent an important source of nutrients for plants. Biochemical analysis demonstrated an increase of enzyme activity, especially H+-ATPase in 2 mM C L-1 landfill humic acid. Additionally, cytogenetic alterations were observed in meristematic and F1 cells, through nuclear abnormalities and micronuclei. Multivariate statistical analysis provided integration of physical, chemical and biological data. Despite all the nutritional benefits of humic acids and their activation of plant antioxidant systems, the observed biological effects showed concerning levels of mutagenicity.

Effects of ammonia stress in the Amazon river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

We evaluate the effects of total ammonia nitrogen-N (TAN) exposure for 72h on (Na(+),K(+))- and V(H(+))-ATPase activities and on their subunit expressions in gills of the diadromous freshwater shrimp Macrobrachium amazonicum. Specific (Na(+),K(+))- and V(H(+))-ATPase activities increased roughly 1.5- to 2-fold, respectively, after exposure to 2.0mmolL(-1) TAN. Quantitative RT-PCR analyses revealed a 2.5-fold increase in V(H(+))-ATPase B subunit mRNA expression while (Na(+),K(+))-ATPase α-subunit expression was unchanged. Immunohistochemical analyses of the gill lamellae located the (Na(+),K(+))-ATPase throughout the intralamellar septal cells, independently of TAN concentration, while the V(H(+))-ATPase was located in both the apical pillar cell flanges and pillar cell bodies. Systemic stress parameters like total hemocyte count decreased by 30% after exposure to 2.0mmolL(-1) TAN, accompanied by increased activities of the oxidative stress enzymes superoxide dismutase, glutathione reductase and glucose-6-phosphate dehydrogenase in the gills. The stress responses of M. amazonicum to elevated TAN include increases in gill (Na(+),K(+))- and V(H(+))-ATPase activities that are accompanied by changes in oxidative stress enzyme activities, immune system effects and an increase in gill V(H(+))-ATPase gene expression. These findings likely underpin physiological effects in a crustacean like M. amazonicum that exploits multiple ecosystems during its life cycle, as well as under culture conditions that may significantly impact shrimp production by the aquaculture industry.

Subchronic exposure to diflubenzuron causes health disorders in neotropical freshwater fish, Prochilodus lineatus.

The action of diflubenzuron (DFB) was evaluated in a freshwater fish, Prochilodus lineatus, after exposure to 0.06, 0.12, 0.25, or 0.50 mg L(-1) DFB for 14 days. Erythrocyte nuclear abnormalities (ENA), the gill activity of Na(+)/K(+)-ATPase, H(+)-ATPase and carbonic anhydrase (CA), and lipid peroxidation (LPO) and histopathological changes in the gills and liver were determined. The number of micronuclei increased in fish exposed to 0.25 and 0.50 mg L(-1) DFB. Plasma Cl(-) and the CA activity decreased, while the activity of Na(+)/K(+)-ATPase and of H(+)-ATPase increased in fish exposed to 0.25 and 0.50 mg L(-1) DFB. LPO did not change in the gills but increased in the liver of fish exposed to 0.25 and 0.50 mg L(-1) DFB. In the gills, histopathological changes indicated disperse lesions and slight to moderate damage in fish exposed to 0.50 mg L(-1) DFB, whereas in the liver, these changes were significantly greater in fish exposed to 0.25 and 0.50 mg L(-1) DFB, indicating moderate to severe damage. Continuous exposure to DFB is potentially toxic to P. lineatus, causing heath disorders when the fish is exposed to the two highest DFB concentrations, which are applied to control parasites in aquaculture and to control mosquito populations in the environment.

A kinetic characterization of the gill V(H(+))-ATPase in juvenile and adult Macrobrachium amazonicum, a diadromous palaemonid shrimp.

Novel kinetic properties of a microsomal gill V(H(+))-ATPase from juvenile and adult Amazon River shrimp, Macrobrachium amazonicum, are described. While protein expression patterns are markedly different, Western blot analysis reveals a sole immunoreactive band, suggesting a single V(H(+))-ATPase subunit isoform, distributed in membrane fractions of similar density in both ontogenetic stages. Immunofluorescence labeling locates the V(H(+))-ATPase in the apical regions of the lamellar pillar cells in both stages in which mRNA expression of the V(H(+))-ATPase B-subunit is identical. Juvenile (36.6±3.3 nmol Pi min(-1) mg(-1)) and adult (41.6±1.3 nmol Pi min(-1) mg(-1)) V(H(+))-ATPase activities are similar, the apparent affinity for ATP of the adult enzyme (K0.5=0.21±0.02 mmol L(-1)) being 3-fold greater than for juveniles (K0.5=0.61±0.01 mmol L(-1)). The K0.5 for Mg(2+) interaction with the juvenile V(H(+))-ATPase (1.40 ± 0.07 mmol L(-1)) is ≈6-fold greater than for adults (0.26±0.02 mmol L(-1)) while the bafilomycin A1 inhibition constant (KI) is 45.0±2.3 nmol L(-1) and 24.2±1.2 nmol L(-1), for juveniles and adults, respectively. Both stages exhibited residual bafilomycin-insensitive ATPase activity of ≈25 nmol Pi min(-1) mg(-1), suggesting the presence of ATPases other than the V(H(+))-ATPase. These differences may reflect a long-term regulatory mechanism of V(H(+))-ATPase activity, and suggest stage-specific enzyme modulation. This is the first kinetic analysis of V(H(+))-ATPase activity in different ontogenetic stages of a freshwater shrimp and allows better comprehension of the biochemical adaptations underpinning the establishment of palaemonid shrimps in fresh water.

Acute effects of cadmium on osmoregulation of the freshwater teleost Prochilodus lineatus: enzymes activity and plasma ions.

Cadmium (Cd) is a trace element that is very toxic to fish. It is commonly found in surface waters contaminated with industrial effluents. When dissolved in water, Cd can rapidly cause physiological changes in the gills and kidneys of freshwater fish. The objective of this study was to evaluate the acute effects of Cd on the osmoregulation of the Neotropical fish Prochilodus lineatus. Juvenile fish were exposed to Cd at two concentrations [1 (Cd1) and 10 (Cd10) µgL(-1)] for 24 and 96h. The effects of Cd were evaluated through the analysis of ions (Na(+), K(+), Ca(2+), and Cl(-)) and plasma osmolality, and by measuring the activities of enzymes involved in osmoregulation obtained from the gills and kidney. Fish exposed to Cd for 24 and 96h showed a decrease in Na(+)/K(+)-ATPase activity in the gills and kidney. The activity of carbonic anhydrase decreased in the gills after 24h and in both tissues after 96h of Cd exposure. The gill Ca(2+)-ATPase activity also decreased with Cd exposure, with a concomitant drop in the plasma concentration of Ca(2+). Despite the hypocalcemia, there were no changes in the concentration of the ions Na(+), K(+), and Cl(-) or in plasma osmolality. Among the enzymes involved in ion transport, H(+)-ATPase was the only enzyme that showed increased activity in gills, whereas its activity in kidney remained unchanged. The results of this study demonstrate that waterborne Cd at the maximum concentrations set by Brazilian guidelines for freshwater affects the gills and kidney functions of P. lineatus. Acute exposure to Cd resulted in the decrease of the activity of enzymes, which culminated with the loss of the fish's ability to regulate the levels of calcium in the blood, leading to hypocalcemia.

Progression of microalbuminuria in SHR is associated with lower expression of critical components of the apical endocytic machinery in the renal proximal tubule.

Cumulative epidemiological evidence indicates that the presence of microalbuminuria predicts a higher frequency of cardiovascular events, peripheral disease, and mortality in essential hypertension. Microalbuminuria may arise from increased glomerular permeability and/or reduced proximal tubular reabsorption of albumin by receptor-mediated endocytosis. This study aimed to evaluate the temporal pattern of urinary protein excretion and to test the hypothesis that progression of microalbuminuria is associated with decreased protein expression of critical components of the endocytic apparatus in the renal proximal tubule of spontaneously hypertensive rats (SHR). We found that urinary albumin excretion increased progressively with blood pressure in SHR from 6 to 21 wk of age. In addition, SDS-PAGE analysis of urinary proteins showed that microalbuminuric SHR virtually excreted proteins of the size of albumin or smaller (<70 kDa), typical of tubular proteinuria. Moreover, the protein abundance of the endocytic receptors megalin and cubilin as well as of the chloride channel ClC-5 progressively decreased in the renal cortex of SHR from 6 to 21 wk of age. Expression of the vacuolar H⁺-ATPase B2 subunit was also reduced in the renal cortex of 21-wk-old compared with both 6- and 14-wk-old SHR. Collectively, our study suggests that enhanced urinary protein excretion, especially of albumin, may be due, at least in part, to lower expression of key components of the apical endocytic apparatus in the renal proximal tubule. Finally, one may speculate that dysfunction of the apical endocytic pathway in the renal proximal tubule may contribute to the development of microalbuminuria in essential hypertension.