Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg+ by (Na+ + K+)-activated ATPase

(1) Contrary to what has usually been assumed, (Na⁺ + K⁺)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na⁺ + Mg²⁺ to ADP-NH₂ and P_i. The activity is ouabain-sensitive and is not detected in the absence of either Mg²⁺ or Na²⁺. The specific activity of the Na⁺ + Mg²⁺ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K⁺. The activity is not stimulated by K⁺, nor can K⁺ replace Na⁺ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na⁺ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The K_m value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH₂]P are not detectably hydrolysed by (Na⁺ + K⁺)-ATPase in the presence of Na⁺ + Mg²⁺. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h^?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg²⁺ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h^?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na⁺ + Mg²⁺ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na⁺ + K⁺)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C.     

CD34+-derived CD11c+ + + BDCA-1+ + CD123+ + DC: expansion of a phenotypically undescribed myeloid DC1 population for use in adoptive immunotherapy

BACKGROUND: DC are commonly defined as HLA-DR+/Lin- cells that can be CD11c+ + + CD123+/ -, termed DC1/myeloid DC that induce a Th1 response, or CD11c- CD123+ + +, termed DC2/lymphoid DC that induce a Th2 response. However, significant heterogeneity within DC preparations is apparent and supports the existence of several distinct DC subpopulations. This study aimed to expand and characterize CD34+ DC for use in immunotherapy. METHODS: CD34+ cells were seeded at 1 x 10(5)/mL and expanded for 14 days in RPMI + 10% autologous plasma supplemented with GM-CSF, IL-4, Flt-3L and SCF. Maturation was induced with TNF-alpha and PGE2 for 2 days. DC were analyzed morphologically, phenotypically with a panel of MAb to lineage and DC markers, and functionally in MLR, T-cell assays and T-cell cytokine secretion by ELISA. RESULTS: Significant cellular expansion was observed: 60+/-5 x 10(6) DC from 1 x 10(6) CD34+ cells (n=28). Phenotypically DC were characterized as HLA-DR+ +, CD11c+ + +, CD80+ +, CD83+, CD86+ +, CD123+ +, CD15+ +, CD33+ +, BDCA-1+ +, CD4+ and Lin-. DC displayed potent allostimulatory capacity and efficient presentation of KLH and tetanus toxin. DC-primed T cells secreted IFN-gamma (Th1); however, no detectable IL-4 (Th2) was noted. DISCUSSION: We present features of CD34+ DC that have not been previously described. The CD34+ DC generated represent a population of myeloid DC functioning as DC1 but phenotypically expressing markers characteristic of both DC1 and DC2. This novel DC population is capable of inducing naive T-cell responses and can be expanded to clinically useful numbers. CD34+-derived DC represent attractive candidates for use in adoptive T-cell immunotherapy.     

Variable coupling of active NA+ + K+ transport in Ehrlich ascites tumor cells: Regulation by external NA+ and K+

The effects of altered external sodium and potassium concentrations on steady state, active Na⁺ + K⁺ transport in Ehrlich ascites tumor cells have been investigated. Membrane permeability to Na⁺ and K⁺, intracellular [Na⁺] and [K⁺], and membrane potential were measured. Active cation fluxes were calculated as equal and membrane potential were measured. Active cation fluxes were calculated as equal and opposite to the net, diffusional leak fluxes. Elevation of external K⁺ (6–60 Mm)by equivalent replacement of Na⁺ (154–91 mM) inhibits both active Na⁺ and K⁺ fluxes, but not proportionally. This results in a decrease of the coupling ratio (r_p = -J_k^p/J) as external K⁺ is increased. Elevation of external K⁺ (3–68 mM) at constant Na⁺ (92mM) inbibits J, but is without effect on J. The coupling ratio declines from 1.01 ± 0.14 to 0.07 ± 0.05, a 14-fold alteration. Reduction of external Na⁺ (154–25 mM) at constant K⁺ (6mM) depresses J, but is without effect on J. The coupling ratio increases from 0.63 ± 0.04 at 154 mM Na⁺ to 4.5 ± 2.04 at 25 mM Na⁺. The results of this investigation are consistent with the independent regulation of active cation fluxes by the transported species. Kinetic analysis of the data indicates that elevation of external sodium stimulates active sodium efflux by interacting at “modifier sites” at the outer cell surface. Similarly, external potassium inhibits active potassium influx by interaction at separate modifier sites.     

Ultrastructure of E1 + 2 + 9 + 12 inversion breakpoints in Drosophila subobscura

The ultrastructure of the Drosophila subobscura chromosome regions around the breakpoints of the complex E1 + 2 + 9 + 12 gene arrangement was analyzed. This overlapping inversion is formed by the association of the E1, E2, E9, and E12 simple inversions. Ultrastructure of sections involving 58D/59A, 61C/D, 62D/63A, 64B/C, 67A/B, and 68B/C breakpoints on Est chromosomes were compared with the ultrastructure of sections involving chromosomes were compared with the ultrastructure of sections involving 58D/68B, 62D/64C, 59A/63A, 64B/68C, 67B/61C, and 67A/61B breakpoints on E1 + 2 + 9 + 12 chromosomes. No detectable changes of structural organization on banding patterns induced by the E1 + 2 + 9 + 12 inversion were found. Ultrastructural analysis of the two E12 breakpoints has, however, facilitated the analysis of the left boundary of E12 inversion. Accordingly, we propose 61B/C as a new breakpoint instead of 61C/D.     

Characterization of Na+K+-ATPase in bovine sperm

Existing as a ubiquitous transmembrane protein, Na⁺K⁺-ATPase affects sperm fertility and capacitation through ion transport and a recently identified signaling function. Functional Na⁺K⁺-ATPase is a dimer of α and β subunits, each with isoforms (four and three, respectively). Since specific isoform pairings and locations may influence or indicate function, the objective of this study was to identify and localize subunits of Na⁺K⁺-ATPase in fresh bull sperm by immunoblotting and immunocytochemistry using antibodies against α1 and 3, and all β isoforms. Relative quantity of Na⁺K⁺-ATPase in head plasma membranes (HPM's) from sperm of different bulls was determined by densitometry of immunoblot bands, and compared to bovine kidney. Sperm and kidney specifically bound all antibodies at kDa equivalent to commercial controls, and to additional lower kDa bands in HPM. Immunofluorescence of intact sperm confirmed that all isoforms were present in the head region of sperm and that α3 was also uniformly distributed post-equatorially. Permeabilization exposing internal membranes typically resulted in an increase in fluorescence, indicating that some antibody binding sites were present on the inner surface of the HPM or the acrosomal membrane. Deglycosylation of β1 reduced the kDa of bands in sperm, rat brain and kidney, with the kDa of the deglycosylated bands differing among tissues. Two-dimensional blots of β1 revealed three distinct spots. Based on the unique quantity, location and structure Na⁺K⁺-ATPase subunits in sperm, we inferred that this protein has unique functions in sperm.     

Ba2+ unmasks K+ modulation of the Na+-K+ pump in the frog retinal pigment epithelium

This paper presents electrophysiological evidence that small changes in [K+]o modulate the activity of the Na+-K+ pump on the apical membrane of the frog retinal pigment epithelium (RPE). This membrane also has a large relative K+ conductance so that lowering [K+]o hyperpolarizes it and therefore increases the transepithelial potential (TEP). Ba2+, a K+ channel blocker, eliminated these normal K+-evoked responses; in their place, lowering [K+]o evoked an apical depolarization and TEP decrease that were blocked by apical ouabain or strophanthidin. These data indicate that Ba2+ blocked the major K+ conductance(s) of the RPE apical membrane and unmasked a slowing of the normally hyperpolarizing electrogenic Na+-K+ pump caused by lowering [K+]o. Evidence is also presented that [K+]o modulates the pump in the isolated RPE under physiological conditions (i.e., without Ba2+). In the intact retina, light decreases subretinal [K+]o and produces the vitreal-positive c-wave of the electroretinogram (ERG) that originates primarily in the RPE from a hyperpolarization of the apical membrane and TEP increase. When Ba2+ was present in the retinal perfusate, the apical membrane depolarized in response to light and the TEP decreased so that the ERG c-wave inverted. The retinal component of the c-wave, slow PIII, was abolished by Ba2+. The effects of Ba2+ were completely reversible. We conclude that Ba2+ unmasks a slowing of the RPE Na+-K+ pump by the light-evoked decrease in [K+]o. Such a response would reduce the amplitude of the normal ERG c-wave.     

Lyt phenotype of cytotoxic T cells: shift from Lyt-1+2+3+ to a mixed population of Lyt-1+2+3+ and Lyt-1-2+3+ during in vitro culture

The Lyt phenotype of cytotoxic T cells generated in the primary H-2 response was investigated kinetically. The cytotoxicity generated in the early stage of culture was abolished by treatment with alpha Lyt-1,2,3, and complement (C), whereas that generated in the late stage was only partially eliminated by alpha Lyt-1, but was abolished by alpha Lyt-2, 3, and C. This suggested late expansion of the Lyt-1-2+3+ population. Lack of Lyt-1 antigen was confirmed with cells that were depleted of Lyt-1+ from primary culture and then stimulated in the secondary response by elimination of cytotoxicity and by direct Lyt typing. Results indicated that the response of proliferative and cytotoxic T cells of the Lyt-1+2+3+ phenotype in the early stage of culture was followed by activation of Lyt-1-2+3+ T cells. Cytotoxic T cells in the late stage were shown to be a mixture of Lyt-1+2+3+ and Lyt-1-2+3+ cells. This was confirmed with cytotoxic T cells from secondary culture and uncloned long-term T cell lines.     

H+- and K+-Dependence of Ca2+ Uptake in Lung Lamellar Bodies

Lung lamellar bodies maintain an acidic interior by an energy-dependent process. The acidic pH may affect the packaging of surfactant phospholipids, processing of surfactant proteins, or surfactant protein A-dependent lipid aggregation. The electron-probe microanalysis of lamellar body elemental composition has previously suggested that lamellar bodies contain high levels of calcium some of which may be in ionic form. In this study, we investigated the Ca²⁺ uptake characteristics in isolated lung lamellar bodies. The uptake of Ca²⁺ was measured by monitoring changes in the fluorescence of Fluo-3, a Ca²⁺ indicator dye. The uptake of Ca²⁺ in lamellar bodies was ATP-dependent and increased with increasing concentrations of Ca²⁺. At 100 nm Ca²⁺, the uptake was almost completely inhibited by bafilomycin A1, a selective inhibitor of vacuolar type H⁺-ATPase, or by NH₄Cl, which raises the lamellar body pH, suggesting that the pH gradient regulates the uptake. The uptake of Ca²⁺ increased as the Ca²⁺ concentration was increased, but the relative contribution of bafilomycin A1-sensitive uptake decreased. At 700 nm, it comprised only 20% of the total uptake. These results suggest the presence of additional mechanism(s) for uptake at higher Ca²⁺ concentrations. At 700 nm Ca²⁺, the rate and extent of uptake were lower in the absence of K⁺ than in the presence of K⁺. The inhibitors of Ca²⁺-activated K⁺-channels, tetraethylammonium, Penitrem A, and 4-aminopyridine, also inhibited the K⁺-dependent Ca²⁺ uptake at 700 nm Ca²⁺. Thus the uptake of Ca²⁺ in isolated lung lamellar bodies appears to be regulated by two mechanisms, (i) the H⁺-gradient and (ii) the K⁺ transport across the lamellar body membrane. We speculate that lamellar bodies accumulate Ca²⁺ and contribute to regulation of cytosolic Ca²⁺ in type II cells under resting and stimulated conditions. Received: 18 August 1999/Revised: 9 November 1999     

K+-Na+ Exchange and Selectivity in Barley Root Cells: Effect of Na+ on the Na+ Fluxes

Using the compartmental analysis the unidirectional Na⁺ fluxesin cortical cells of barley roots, the cytoplasmic and vacuolarNa⁺ contents Q_c and Q_v, and the trans-root Na⁺ transport R'have been studied as a function of the external Na⁺ concentration.Using the re-elution technique the effect of low K⁺ concentrationson the plasmalemma efflux _co of Na⁺ (K+-Na+ exchange) and onR' was investigated at different Na+ concentrations and correspondinglydifferent values of the cytoplasmic sodium content Qc. The relationof the K+-dependent Na+ efflux coK+-dep to Qc or to the cytoplasmicNa+ concentration obeyed Michaelis-Menten kinetics. This isconsistent with a linkage of co, K+-dep to K+ influx by a K⁺-Na⁺exchange system. The apparent Km corresponded to a cytoplasmicNa⁺ concentration of 28 mM at 0·2 mM K⁺ and about 0·2mM Na⁺ in the external solution. 0·2 mM K⁺ stimulatedthe plasma-lemma efflux of Na⁺ and inhibited Na⁺ transport selectivelyeven in the presence of 10 mM Na⁺ in the external medium showingthe high efficiency of the K⁺-Na⁺ exchange system. However,_co, K⁺-dep was inhibited at 10 mM Na¹ compared to lower Na¹concentrations suggesting some competition of Na¹ with K¹ atthe external site of the exchange system. The effect of theNa+ concentration on Na¹ influx _oc is discussed with respectto kinetic models of uuptake.     

Sidedness of the ATP-Na+-K+ interactions with the Na+ pump in squid axons

Using dialysed squid axons we have been able to control internal and external ionic compositions under conditions in which most of the Na⁺ efflux goes through the Na⁺ pump. We found that (i) internal K⁺ had a strong inhibitory effect on Na⁺ efflux; this effect was antagonized by ATP, with low affinity, and by internal Na⁺, (ii) a reduction in ATP levels from 3 mM to 50 μM greatly increased the apparent affinity for external K⁺, but reduced its effectiveness compared with other monovalent cations, as an activator of Na⁺ efflux, and (iii) the relative effectiveness of different K⁺ congeners as external activator of the Na⁺ efflux, though affected by the ATP concentration, was not affected by the Na⁺/⁺ ratio inside the cells. These results are consistent with the idea that the same conformation of the (Na⁺ + K⁺)-ATPase can be reached by interaction with external K⁺ after phosphorylation and with internal K⁺ before rephosphorylation. They also stress a nonphosphorylating regulatory role of ATP.     

Characterization of the T3+T4+T8+ thymocyte intermediate in vitro

Sensitive two-color fluorescence staining and cell-sorting techniques were used to isolate a T4+T8+ thymocyte subpopulation with high T3 density from human thymocyte cultures. Previously, this population was shown to give rise to both T4+T8- and T8+T4- thymocytes. In the present study, this T3+T4+T8 population was shown to be functionally as well as phenotypically distinct from either T8+T4- cells or T4+T8- cells present in the same culture. The T3+T4+T8+ cell had intermediate cytotoxic capacities relative to T8+T4- and T4+T8- thymocyte fractions. The proliferative capacity of the T4+T8+ population although less than that of the T8+T4- subset exceeded the proliferative response of the T4+T8- population. The time of appearance of large numbers of T3+T4+T8+ cells in culture as well as functional properties exhibited by T3+T4+T8+ cells are consistent with the notion that the T3+T4+T8+ cell represents an activated intermediate in thymocyte differentiation. The T3+T4+T8+ thymocyte may be an important intermediate in in vivo as well as in in vitro thymic differentiation. Moreover, the analysis of its functional properties may contribute to an understanding of functional responses exhibited by the most mature (T3+) population isolated from human thymus.     

Interaction of Sr2+ with Ca2+-induced Ca2+ release in mitochondria

Respiring rat liver mitochondria are known to spontaneously release the Ca²⁺ taken up when they have accumulated Ca²⁺ over a certain threshold, while Sr²⁺ and Mn²⁺ are well tolerated and retained. We have studied the interaction of Sr²⁺ with Ca²⁺ release. When Sr²⁺ was added to respiring mitochondria simultaneously with or soon after the addition of Ca²⁺, the release was potently inhibited or reversed. On the other hand, when Sr²⁺ was added before Ca²⁺, the release was stimulated. Ca²⁺-induced mitochondrial damage and release of accumulated Ca²⁺ is generally believed to be due to activation of mitochondrial phospholipase A (EC 3.1.1.4.) by Ca²⁺. However, isolated mitochondrial phospholipase A activity was little if at all inhibited by Sr²⁺. The Ca²⁺ -release may thus be triggered by some Ca²⁺ -dependent function other than phospholipase.