Ontogeny of the antennal glands in the crayfish Astacus leptodactylus (Crustacea, Decapoda): immunolocalization of Na+,K+-ATPase

The involvement of the antennal urinary glands in the ontogeny of osmoregulatory functions was investigated during the development of Astacus leptodactylus by measurements of hemolymph and urine osmolality in juvenile and adult crayfish and by the immunodetection of the enzyme Na⁺,K⁺-ATPase. In stage II juveniles, 1-year-old juveniles, and adults, all of which were maintained in freshwater, urine was significantly hypotonic to hemolymph. In adults, chloride and sodium concentrations were much lower in urine than in hemolymph. During embryonic development, Na⁺,K⁺-ATPase was detected by immunocytochemistry in ionocytes lining the tubule and the bladder, at an eye index (EI) of 220–250 m, and in the labyrinth, at EI 350 m. In all regions, immunofluorescence was mainly located at the basolateral side of the cells. No immunofluorescence was detected at any stage in the coelomosac. In late embryonic stages (EI 410–440 m), in stage I juveniles, and in adults, strong positive immunofluorescence was found from the labyrinth up to and including the bladder. These results show that, as early as hatching, juvenile crayfish are able to produce dilute urine hypotonic to hemolymph. This ability originates from the presence of Na⁺,K⁺-ATPase in ion-transporting cells located in the labyrinth, the tubule, and the bladder of the antennal glands and constitutes one of the main adaptations of crayfish to freshwater.We thank the University of Tarbiat Modarres and Ministry of Science, Research and Technology, Islamic Republic of Iran for financial aid and support. Special thanks are also due to the Société Française dExportation des Ressources Educatives (SFERE) for the scholarship to S.K.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Antennal thermo- and hygrosensitive sensilla in Antheraea pernyi (Lepidoptera,Saturniidae): ultrastructure and immunohistochemical localization of Na+,K+-ATPase

Summary In an attempt to identify and localize the components of voltage sources involved in sensory transduction in insect sensilla, the thermo-/hygrosensitive sensilla of the moth Antheraea pernyi were probed with a polyclonal antiserum against Na⁺,K⁺-ATPase in cryofixed and freeze-substituted preparations. The antiserum recognized epitopes on the cytoplasmic membranes of the dendritic inner segments and somata of the sensory cells and also on the cytoplasmic membranes of glial cells surrounding the initial axon segments. The findings support the current concept that ion pumps in the cytoplasmic membranes of the dendritic inner segments and somata of the sensory cells contribute to the maintenance of the resting potential of the sensory cells and to the driving forces generating the receptor currents in response to stimulation of the sensillum. Morphological features and immunohistochemical characteristics of the region of the initial axon segment are also discussed with respect to the initiation of action potentials in these sensilla.     

Emission analysis of Tb3+‐and Sm3+‐ion‐doped (Li2O/Na2O/K2O) and (Li2O + Na2O/Li2O + K2O/K2O + Na2O)‐modified borosilicate glasses

Four series of borosilicate glasses modified by alkali oxides and doped with Tb³⁺ and Sm³⁺ ions were prepared using the conventional melt quenching technique, with the chemical composition 74.5B₂O₃ + 10SiO₂ + 5MgO + R + 0.5(Tb₂O₃/Sm₂O₃) [where R = 10(Li₂O /Na₂O/K₂O) for series A and C, and R = 5(Li₂O + Na₂O/Li₂O + K₂O/K₂O + Na₂O) for series B and D]. The X‐ray diffraction (XRD) patterns of all the prepared glasses indicate their amorphous nature. The spectroscopic properties of the prepared glasses were studied by optical absorption analysis, photoluminescence excitation (PLE) and photoluminescence (PL) analysis. A green emission corresponding to the ⁵D₄ → ⁷F₅ (543 nm) transition of the Tb³⁺ ions was registered under excitation at 379 nm for series A and B glasses. The emission spectra of the Sm³⁺ ions with the series C and D glasses showed strong reddish‐orange emission at 600 nm (⁴G_5/2 →⁶H_7/2) with an excitation wavelength λ_exci = 404 nm (⁶H_5/2→⁴F_7/2). Furthermore, the change in the luminescence intensity with the addition of an alkali oxide and combinations of these alkali oxides to borosilicate glasses doped with Tb³⁺ and Sm³⁺ ions was studied to optimize the potential alkali‐oxide‐modified borosilicate glass.     

Hypericin-induced phototoxicity of human leukemic cell line HL-60 is potentiated by omeprazole, an inhibitor of H+K+-ATPase and 5'-(N,N-dimethyl)-amiloride, an inhibitor of Na+/H+ exchanger.

Hypericin, an antiretroviral and antineoplastic agent, seems to have multiple modes of light-induced biological activity connected with the production of single oxygen and/or excited-state proton transfer and a consequent pH drop of pH formation in the hypericin environment. In the present study omeprazole, an inhibitor of H+K+-ATPase, and amiloride, an inhibitor of the Na+/H+ exchanger, have been used for testing the hypothetical pH decreasing effect of hypericin in its antineoplastic action. The results of our experiments have shown that in the HL-60 cell line the effect of hypericin (10(-6) mol.l(-1)) was significantly potentiated by omeprazole and 5'-(N,N-dimethyl)-amiloride. The effect of omeprazole seemed to be less specific than that of 5'-(N,N-dimethyl)-amiloride. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of the hypericin environment could play a role in the biological activity of hypericin. Moreover, both omeprazole and 5'-(N,N-dimethyl)-amiloride are effective potentiating agents of hypericin cytotoxic effect in the HL60 cell line.     

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: Effects on gill (Na,K)-ATPase α-subunit expression and K-phosphatase activity

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21‰ salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6 ± 4.9 U mg^− 1 and K_0.5 = 1.31 ± 0.05 mmol L^− 1. Stimulation of K⁺-phosphatase activity by magnesium (V_max = 125.3 ± 7.5 U mg^− 1; K_0.5 = 2.09 ± 0.06 mmol L^− 1), potassium (V_max = 134.2 ± 6.7 U mg^− 1; K_0.5 = 1.33 ± 0.06 mmol L^− 1) and ammonium ions (V_max = 130.1 ± 5.9 U mg^− 1; K_0.5 = 11.4 ± 0.5 mmol L^− 1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (K_I = 304.9 ± 18.3 μmol L^− 1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the ≈2-fold reduction in K⁺-phosphatase specific activity, Western blotting analysis revealed similar α-subunit expression in gill tissue from shrimps acclimated to 21‰ salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na⁺,K⁺)-ATPase was stimulated by high salinity acclimation.     

Competitive binding of ATP and the fluorescent substrate analogue 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidine) adenosine 5'-triphosphate to the gastric H+,K+-ATPase: evidence for two classes of nucleotide sites

ATP and the fluorescent substrate analogue TNP-ATP bind competitively to the gastric H,-K-ATPase. Substrate and product completely reverse the fluorescence enhancement caused by TNP-ATP binding to the enzyme. The fluorophore is displaced monophasically from apoenzyme. However, ATP displaces TNP-ATP from the Mg2+-quenched state in two steps of equal amplitude. The midpoints of the titrations differ by more than 2 orders of magnitude. The estimated substrate constants are in reasonable agreement with published Michaelis constants. TNP-ATP is not a substrate for the H,K-ATPase. The fluorophore prevents phosphorylation by ATP and competitively inhibits the K+-stimulated pNPPase and ATPase activities of the enzyme. Ki is approximately the same for both hydrolytic activities and consistent with the Kd of TNP-ATP measured directly. Km for pNPP is 1.48 +/- 0.15 mM. Two Michaelis constants are required to fit the ATPase data: Km1 = 0.10 +/- 0.01 mM and Km2 = 0.26 +/- 0.05 mM.     

Sol–gel synthesis and luminescence property of Sr4Al2O7:Re3+,R+ (Re = Eu and Dy; R = Li,Na and K) phosphors for white LEDs

Sr₄Al₂O₇:Eu³⁺ and Sr₄Al₂O₇:Dy³⁺ phosphors with alkali metal substitution were prepared using a sol–gel method. The effects of a charge compensator R on the structure and luminescence of Sr₄Al₂O₇:Re³⁺,R⁺ (Re = Eu and Dy; R = Li, Na and K) phosphors were investigated in detail. Upon heating to 1400°C, the structure of the prepared samples was that of the standard phase of Sr₄Al₂O₇. Under ultraviolet excitation, all Sr₄Al₂O₇:Eu³⁺,R⁺ samples exhibited several narrow emission peaks ranging from 550 to 700 nm due to the 4f → 4f transition of Eu³⁺ ions. All Sr₄Al₂O₇:Dy³⁺,R⁺ phosphors showed two emission peaks at 492 and 582 nm, due to the ⁴F_9/2 → ⁶H_15/2 and ⁴F_9/2 → ⁶H_13/2 transitions of Dy³⁺ ions, respectively. The luminescence intensity of Sr₄Al₂O₇:Re³⁺,R⁺ (Re = Eu and Dy; R = Li, Na and K) phosphors improved markedly upon the addition of charge compensators, promoting their application in white light‐emitting diodes with a near‐ultraviolet chip.