Influence of Bcl-2 overexpression on Na+/K+-ATPase pump activity: Correlation with radiation-induced programmed cell death

Bcl-2 overexpression in transfected PW cells is associated with inhibition of radiation-induced programmed cell death (PCD). We have previously reported that there is a relationship between inhibition of radiation-induced PCD and membrane hyperpolarization in these cells. In this article, we report that Na⁺/K⁺-ATPase pump activity, as measured by the uptake of Rubidium-86 (⁸⁶Rb⁺), is significantly higher in Bcl-2 overexpressing PW cells than in control PW cells, and that pump activity following irradiation with doses ≥ 500 cGy was reduced to a lesser extent in the Bcl-2 transfectants than in the control cells. When PW-Bcl-2 cells were incubated with a dose of ouabain (1 μM) that decreased pump activity significantly, but did not induce PCD, the previously reported protection from radiation-induced PCD associated with overexpression of Bcl-2 no longer existed. In order to demonstrate that reactive oxygen species (ROS) affected Na⁺/K⁺-ATPase pump activity, cells were incubated with N-acetyl cysteine (NAC) prior to irradiation, or treated with the ROS generating drug buthionine sulphoxamine (BSO). ⁸⁶Rb⁺uptake was significantly higher in irradiated cells incubated with NAC compared to cells irradiated in the absence of NAC, while BSO resulted in lower levels of ⁸⁶Rb⁺uptake, suggesting that the effects of radiation on the Na⁺/K⁺-ATPase pump were due to ROS. Furthermore, the resting cell membrane potential of cells exposed to NAC were slightly hyperpolarized compared to control PW cells, whereas cells exposed to BSO were depolarized in comparison to control PW cells. In summary, this data suggests that Bcl-2 affects Na⁺/K⁺-ATPase pump activity, which is associated with the resting membrane potential and the level of susceptibility to radiation-induced PCD. J. Cell. Physiol. 171:299–304, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of Na+, K+-ATPase in Cultured and Separated Neuronal and Glial Cells from Rat Cerebellum

Abstract: The activities of certain properties of sodium, potassium-activated adenosine triphosphatase (Na ⁺, K⁺- ATPase; EC 3.6.1.3) were examined in cultures and peri- karya fractions enriched in rat cerebellar nerve cells or astrocytes, in comparison with preparations from whole immature and adult rat cerebellum and derived synapto- somal fractions, as well as nonneural tissue such as the kidney. The specific activity of Na ⁺, K⁺-ATPase was markedly higher in the freshly isolated astrocytes than in the nerve cells (3–15-fold greater depending on neuronal cell type). In contrast, the specific activity of the enzyme was about twice as high in the primary neuronal as in the a'strocytic cultures after 14 days in vitro. In membrane preparations from the whole cerebellum, synaptosomal fractions, and total perikarya suspensions the inhibition of enzyme activity by ouabain indicated complex kinetics, which were consistent with the presence of two forms of the Na ⁺, K⁺-ATPase (apparent A_j values of about 10–⁷M and 10–⁴-10–⁵M, respectively), the high- affinity form accounting for 60–75% of the total activity. The interaction of the enzyme with ouabain was apparently similar in perikarya preparations of granule neurones, Purkinje cells, and astrocytes. Differences were, however, observed in the properties of the Na ⁺,K ⁺ - ATPase of cultured neurones and astrocytes. The latter contained predominantly, but not exclusively, an Na⁺,K⁺-ATPase with low affinity for ouabain (73% of the total) that is similar to the single enzyme form in the kidney. This form constituted a significantly smaller proportion of the Na ⁺, K⁺-ATPase in the cultured neuronal preparations (55%). It would appear, therefore, that in membrane fractions from preparations enriched in different separated and cultured neural cell types both the high- and the low-affinity forms of the enzyme, in terms of interaction with ouabain, are expressed. Depending on the class of cells these enzyme forms constituted a different proportion of the total activity, but both forms seemed to be present in every type of cell examined, even after taking into acc.ount the contribution in the enriched preparations of the contaminating cell types. In contrast with the results on the Na⁺, K⁺-ATPase activity determined under optimal conditions in preparations derived from disrupted cells, differences could not be detected between the cultured cell types when the effect of ouabain on the uptake of ⁸⁶Rb into “live cells” was estimated as a measure of in situ ion pump activity. Besides the interaction with ouabain, the K⁺ dependence of the Na⁺, K⁺-ATPase activity was also investigated in crude particulate preparations from cultured cerebellar neurones and astrocytes. Differences were observed as nearly maximal enzyme activity was obtained in the as- trocyte preparations at 1 mM KCl, when only about one- third of the maximal activity was displayed by the cultured nerve cells.     

Membrane potentials,electrolyte contents,cell pH,and some enzyme activities of fibroblasts

Summary The resting membrane potential of the cultured fibroblasts derived from rabbit subcutaneous tissues was −10.2±0.20 mV (n=390). This potential was affected by the potassium concentration in the culture medium, but not by other chemical or hormonal preparations, such as dibutyryladenosine 3′,5′-cyclic monophosphate (0.5 to 5.0 mmol/l), sodium fluoride (10⁻⁵ to 10⁻⁴ M), hydrocortisone (10⁻⁷ to 10⁻⁶ M), parathyroid extract (0.5 to 1.0 U/ml), or thyrotrophin (5 to 10 mU/ml). The Na⁺, K⁺, and Cl⁻ concentrations of the cultured fibroblasts were 35.4, 85.7, and 22.6 mmol/l cell water, respectively. The water and protein contents of these cells were 82.1 and 9.18 g/100-g cells, respectively. The intracellular pH of fibroblasts as determined by [¹⁴C] dimethyloxazolidine-2, 4-dione, and³H₂O ranged between 6.9 and 7.1 when the pH of the culture medium was maintained at 7.4. The activiities of Na⁺, K⁺-, HCO₃ ⁻-, and Ca⁺⁺, Mg⁺⁺-ATPases in these cultured cells were 19.0±2.1, 13.6±2.1, and 6.6±1.2 nmol pi/mg protein per minute, respectively, and the carbonic anhydrase activity was 0.054 U/mg protein. Calculations based on the values for the membrane potential and the electrolyte concentrations observed in this study indicate that Na⁺, K⁺, Cl⁻, and H⁺ are not distributed according to their electrochemical gradients across the cell membrane. Na⁺, Cl⁻, and H⁺ are actively transported out of the cells and K⁺ into the cells. This study was supported by Grant AM20935 from the NIAMDD, NIH, Bethesda, Maryland, and National Aeronautics & Space Administration NASA-Ames Grant NAG 2-108 and U.S. Department of Energy Contract DE-AC02-76-EV-00119. D. M. W. is the recipient of a Research Career Award (5-K6-NB-13838), NINCDS, NIH.     

Muscarinic Activation of BK Channels Induces Membrane Oscillations in Glioma Cells and Leads to Inhibition of Cell Migration

Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca²⁺] _i ) associated with periodic Ca²⁺ oscillations during prolonged ACh applications. The early transient rise in [Ca²⁺] _i was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly dependent on external calcium. The Ca²⁺ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine. Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca²⁺] _i oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca²⁺-sensitive K⁺ currents. These K⁺ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin, but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca²⁺ from intracellular stores which in turn activate Ca²⁺-dependent (BK-type) K⁺ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway with glioma cell migration. Received: 17 December/Revised: 17 March 2000     

Passive Glial Cells, Fact or Artifact?

Astrocytes that are recorded in acute tissue slices of rat hippocampus using whole-cell patch-clamp, commonly exhibit voltage-activated Na⁺ and K⁺ currents. Some reports have described astrocytes that appear to lack voltage-activated currents and proposed that these cells constitute a subpopulation of electrophysiologically passive astrocytes. We show here that these cells can spontaneously change during a recording unmasking expression of previously suppressed voltage-activated currents, suggesting that such cells do not represent a subpopulation of passive astrocytes. Superfusion of a low Ca²⁺/EGTA solution was able to reversibly suppress voltage-activated K⁺ currents in cultured astrocytes. Currents were restored upon addition of normal bath Ca²⁺. These effects of Ca²⁺ on both outward and inward K⁺ currents were dose- and time-dependent, with increasing concentrations of Ca²⁺ (from 0 to 800 μm) leading to a gradual unmasking of voltage-dependent outward and inward K⁺ currents. The transition from an apparently passive cell to one exhibiting prominent voltage-activated currents was not associated with any changes in membrane capacitance or access resistance. By contrast, in cells in which low access resistance or poor seal accounted for the absence of voltage-activated currents, improvement of cell access was always accompanied by changes in series resistance and membrane capacitance. We propose that spillage of pipette solution containing low Ca²⁺/EGTA during cell approach in slice recordings and/or poor cell access, lead to a transient masking of voltage-activated currents even in astrocytes that express prominent voltage-activated currents. These cells, however, do not constitute a subpopulation of electrophysiologically passive astrocytes. Received: 22 April 1998/Revised: 8 September 1998     

Osteoblast lineage cells expressing high levels of Runx2 enhance hematopoietic progenitor cell proliferation and function

Although osteoblasts (OB) play a key role in the hematopoietic stem cell (HSC) niche, little is known as to which specific OB lineage cells are critical for the enhancement of stem and progenitor cell function. Unlike hematopoietic cells, OB cell surface phenotypic definitions are not well developed. Therefore, to determine which OB lineage cells are most important for hematopoietic progenitor cell (HPC) function, we characterized OB differentiation by gene expression and OB function, and determined whether associations existed between OB and HPC properties. OB were harvested from murine calvariae, used immediately (fresh OB) or cultured for 1, 2, or 3 weeks prior to their co‐culture with Lin^?Sca1⁺c‐kit⁺ (LSK) cells for 1 week. OB gene expression, alkaline phosphatase activity, calcium deposition, hematopoietic cell number fold increase, CFU fold increase, and fold increase of Lin^?Sca1⁺ cells were determined. As expected, HPC properties were enhanced when LSK cells were cultured with OB compared to being cultured alone. Initial alkaline phosphatase and calcium deposition levels were significantly and inversely associated with an increase in the number of LSK progeny. Final calcium deposition levels and OB culture duration were inversely associated with all HPC parameters, while Runx2 levels were positively associated with all HPC properties. Since calcium deposition is associated with OB maturation and high levels of Runx2 are associated with less mature OB lineage cells, these results suggest that less mature OB better promote HPC proliferation and function than do more mature OB. J. Cell. Biochem. 111: 284–294, 2010. © 2010 Wiley‐Liss, Inc.     

Membrane Potential Measurements in Cultured Intestinal Villi

Fragmented epithelia of newborn rat small intestine were successfully cultured for periods of up to 4 weeks. Stable intracellular recordings of membrane potential were obtained from these cultured cells. Membrane resting potential varied according to cell location along a villus. The potentials ranged from -70 to -15 mV, being highest at the tip of the villus. The mean resting potential and membrane resistance were -72.4 mV and 8.6 M Ω, respectively. The membrane potential was markedly dependent on the extracellular K⁺ concentration ([K]₀], but not significantly on [Na]₀ and [Cl]₀-Deprivation of Ca²⁺ from the surrounding medium depolarized the membrane by 20 mV. When the cells were cooled down to 6°C, membrane potential was reduced by 40 mV. Based on these data, basic mechanisms underlying the resting potential are discussed in connection with cell differentiation or maturation.     

EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inject effector proteins into host intestinal epithelial cells causing diarrhoea. EPEC infection redistributes basolateral proteins β1‐integrin and Na⁺/K⁺ ATPase to the apical membrane of host cells. The Crumbs (Crb) polarity complex (Crb3/Pals1/Patj) is essential for epithelial cell polarisation and tight junction (TJ) assembly. Here, we demonstrate that EPEC displaces Crb3 and Pals1 from the apical membrane to the cytoplasm of cultured intestinal epithelial cells and colonocytes of infected mice. In vitro studies show that EspF, but not Map, alters Crb3, whereas both effectors modulate Pals1. EspF perturbs polarity formation in cyst morphogenesis assays and induces endocytosis and apical redistribution of Na⁺/K⁺ ATPase. EspF binds to sorting nexin 9 (SNX9) causing membrane remodelling in host cells. Infection with ΔespF/pespFD3, a mutant strain that ablates EspF binding to SNX9, or inhibition of dynamin, attenuates Crb3 endocytosis caused by EPEC. In addition, infection with ΔespF/pespFD3 has no impact on Na⁺/K⁺ ATPase endocytosis. These data support the hypothesis that EPEC perturbs apical–basal polarity in an EspF‐dependent manner, which would contribute to EPEC‐associated diarrhoea by disruption of TJ and altering the crucial positioning of membrane transporters involved in the absorption of ions and solutes.     

Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

Autoimmunity is associated with defective phagocytic clearance of apoptotic cells. IgM deficient mice exhibit an autoimmune phenotype consistent with a role for circulating IgM antibodies in apoptotic cell clearance. We have extensively characterised IgM binding to non-apoptotic and apoptotic mouse thymocytes and human Jurkat cells using flow cytometry, confocal imaging and electron microscopy. We demonstrate strong specific IgM binding to a subset of Annexin-V (AnnV)⁺PI (Propidium Iodide)⁺ apoptotic cells with disrupted cell membranes. Electron microscopy studies indicated that IgM⁺AnnV⁺PI⁺ apoptotic cells exhibited morphologically advanced apoptosis with marked plasma membrane disruption compared to IgM^-AnnV⁺PI⁺ apoptotic cells, suggesting that access to intracellular epitopes is required for IgM to bind. Strong and comparable binding of IgM to permeabilised non-apoptotic and apoptotic cells suggests that IgM bound epitopes are ''apoptosis independent'' such that IgM may bind any cell with profound disruption of cell plasma membrane integrity. In addition, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognize and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption. The role of IgM in vivo in conditions characterized by severe cell damage such as ischemic injury, sepsis and thrombotic microangiopathies merits further exploration.     

C-peptide,Na+,K+-ATPase,and Diabetes

Na⁺,K⁺-ATPase is an ubiquitous membrane enzyme that allows the extrusion of three sodium ions from the cell and two potassium ions from the extracellular fluid. Its activity is decreased in many tissues of streptozotocin-induced diabetic animals. This impairment could be at least partly responsible for the development of diabetic complications. Na⁺,K⁺-ATPase activity is decreased in the red blood cell membranes of type 1 diabetic individuals, irrespective of the degree of diabetic control. It is less impaired or even normal in those of type 2 diabetic patients. The authors have shown that in the red blood cells of type 2 diabetic patients, Na⁺,K⁺-ATPase activity was strongly related to blood C-peptide levels in non–insulin-treated patients (in whom C-peptide concentration reflects that of insulin) as well as in insulin-treated patients. Furthermore, a gene-environment relationship has been observed. The alpha-1 isoform of the enzyme predominant in red blood cells and nerve tissue is encoded by the ATP1A1 gene.Apolymorphism in the intron 1 of this gene is associated with lower enzyme activity in patients with C-peptide deficiency either with type 1 or type 2 diabetes, but not in normal individuals. There are several lines of evidence for a low C-peptide level being responsible for low Na⁺,K⁺-ATPase activity in the red blood cells. Short-term C-peptide infusion to type 1 diabetic patients restores normal Na⁺,K⁺-ATPase activity. Islet transplantation, which restores endogenous C-peptide secretion, enhances Na⁺,K⁺-ATPase activity proportionally to the rise in C-peptide. This C-peptide effect is not indirect. In fact, incubation of diabetic red blood cells with C-peptide at physiological concentration leads to an increase of Na⁺,K⁺-ATPase activity. In isolated proximal tubules of rats or in the medullary thick ascending limb of the kidney, C-peptide stimulates in a dose-dependent manner Na⁺,K⁺-ATPase activity. This impairment in Na⁺,K⁺-ATPase activity, mainly secondary to the lack of C-peptide, plays probably a role in the development of diabetic complications. Arguments have been developed showing that the diabetesinduced decrease in Na⁺,K⁺-ATPase activity compromises microvascular blood flow by two mechanisms: by affecting microvascular regulation and by decreasing red blood cell deformability, which leads to an increase in blood viscosity. C-peptide infusion restores red blood cell deformability and microvascular blood flow concomitantly with Na⁺,K⁺-ATPase activity. The defect in ATPase is strongly related to diabetic neuropathy. Patients with neuropathy have lower ATPase activity than those without. The diabetes-induced impairment in Na⁺,K⁺-ATPase activity is identical in red blood cells and neural tissue. Red blood cell ATPase activity is related to nerve conduction velocity in the peroneal and the tibial nerve of diabetic patients. C-peptide infusion to diabetic rats increases endoneural ATPase activity in rat. Because the defect in Na⁺,K⁺-ATPase activity is also probably involved in the development of diabetic nephropathy and cardiomyopathy, physiological C-peptide infusion could be beneficial for the prevention of diabetic complications.     

Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells

Summary For patch-clamp measurements cultured kidney (OK) cells were exposed to osmotic and mechanical stress. Superfusion of a cell in whole cell configuration with hypotonic media (190 mOsm) evokes strong depolarization, which is reversible by returning to the isotonic bath medium. In the cell-attached configuration the exposure to hypotonic media evokes up to six ion channels of homogeneous single-channel properties in the membrane patch. Subsequently, the channels became activated after a time lag of a few seconds. At an applied membrane potential of 0 mV, the corresponding membrane current is directed inward and shows a transient behavior in the time range of minutes. In the same membrane patch these ion channels can be activated by application of negative hydrostatic pressure. The channel has a single-channel conductance of about 22 pS and is permeable to Na⁺ and K⁺ as well as to Cl^–. It is suggested that volume regulation involves mechanoreceptor-operated ion channels.     

The Basis of Higher Na+ Transport by Inner Medullary Collecting Duct Cells from Dahl Salt-Sensitive Rats: Implicating the Apical Membrane Na+ Channel

The present experiments were designed to examine the function of Na/K pumps from Dahl salt-sensitive (S) and salt-resistant (R) rats. Previous reports have suggested that there is a difference in primary sequence in the α₁ subunit, the major Na/K pump isoform in the kidney. This sequence difference might contribute to differences in NaCl excretion in these two strains which in turn could influence the systemic blood pressure. Using ``back-door' phosphorylation of pumps isolated from basolateral membranes of kidney cortex, we found no differences between S and R strains. We also examined the Na/K pumps from cultured inner medullary collecting duct (IMCD) cells. This approach takes advantage of the fact that monolayers cultured from S rats transport about twice as much Na⁺ as monolayers cultured from R rats. In cells whose apical membrane was made permeable with amphotericin B, comparison of the affinities for ouabain, Na⁺, and K⁺, respectively, showed only small or no differences between S and R monolayers. Ouabain binding showed no difference in the number of Na/K pumps on the basolateral membrane of cultured cells, despite a 2-fold difference in Na⁺ transport rates. The analysis of the steady-state Na⁺ transport indicates that Na/K pumps in IMCD monolayers from S rats operate at a higher fraction of their maximum capacity than do pumps in monolayers from R rats. The results, taken together, suggest that the major reason for the higher rate of Na⁺ transport in S monolayers is because of a primary increase in the conductive permeability of the apical membrane to Na⁺. They suggest that the epithelial Na⁺ channel is intrinsically different or differently regulated in S and R rats. Received: 6 May 1996/Revised: 16 October 1996     

Tubulin–Na+ ,K +-ATPase interaction: Involvement in enzymatic regulation and cellular function

A new function for tubulin was described by our laboratory: acetylated tubulin forms a complex with Na⁺,K ⁺-ATPase (NKA) and inhibits its activity. This process was shown to be a regulatory factor of physiological importance in cultured cells, human erythrocytes, and several rat tissues. Formation of the acetylated tubulin–NKA complex is reversible. We demonstrated that in cultured cells, high concentrations of glucose induce translocation of acetylated tubulin from cytoplasm to plasma membrane with a consequent inhibition of NKA activity. This effect is reversed by adding glutamate, which is coctransported to the cell with Na ⁺. Another posttranslational modification of tubulin, detyrosinated tubulin, is also involved in the regulation of NKA activity: it enhances the NKA inhibition induced by acetylated tubulin. Manipulation of the content of these modifications of tubulin could work as a new strategy to maintain homeostasis of Na ⁺ and K ⁺, and to regulate a variety of functions in which NKA is involved, such as osmotic fragility and deformability of human erythrocytes. The results summarized in this review show that the interaction between tubulin and NKA plays an important role in cellular physiology, both in the regulation of Na ⁺/K ⁺ homeostasis and in the rheological properties of the cells, which is mechanically different from other roles reported up to now.     

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na⁺,K⁺-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H⁺,K⁺-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb⁺ or Li⁺ transport by Na⁺,K⁺- or gastric H⁺,K⁺-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb⁺ (Li⁺) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb⁺ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na⁺/2K⁺ transport stoichiometry of the Na⁺,K⁺-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H⁺,K⁺-ATPase. In principle, the assay is not limited to K⁺-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.     

Control of normal differentiation of myeloid leukemic cells. X. Glucose utilization,cellular ATP and associated membrane changes in D+ and D− cells

Glucose utilization, energy metabolism and associated membrane changes, have been studied in D⁺ myeloid leukemic cells that can be induced to undergo cell differentiation to mature granulocytes by incubation with the appropriate conditioned medium (CM) and in D^? myeloid leukemic cells that cannot be induced to differentiate to mature cells. Before incubation with CM, glycolysis and the glycolytic production of ATP were lower and the activity of the pentose cycle was higher in D⁺ than in D^? cells. ATP depletion induced a higher degree of agglutination by concanavalin A in D^? than in D⁺ cells, indicating a difference in their surface membrane. There were no detectable differences in the transport of glucose and the synthesis of sterols and fatty acids. After incubation with CM, the D⁺ cells, like normal granulocytes, showed a higher glycolysis, produced their ATP more through glycolysis than oxidative phosphorylation, became less dependent on the exogenous supply of glucose and oxygen and had a lower rate of sterol and fatty acid synthesis. The differentiating D⁺ cells also showed a change in their surface membrane resulting in an increased agglutinability without a change in ATP content and a stimulation of the pentose cycle by concanavalin A. These properties, which were not acquired by D^? cells, were found before most of the D⁺ cells had differentiated to mature granulocytes. The data indicate, that the block in the ability of the D^? cells to differentiate and the acquisition of the metabolic properties of normal granulocytes by differentiating D⁺ cells, were associated with differences in the organization of the cell surface membrane.     

Mechanism of high K+ and Tl+ uptake in cultured human glioma cells

Summary 1. The aim of this study was to elucidate if the K⁺ uptake was higher in cultured human glioma cells than in cells from other malignant tumors and to analyze the importance of membrane potential and K⁺ channels for the uptake.2. K⁺ transport properties were studied with the isotopes⁴²K and the K-analogue²⁰¹Tl.3. Comparison with cultured cells from other malignant tumors showed that the specific steady-state accumulation of Tl⁺ was significantly higher in glioma cells (U-251MG and Tp-378MG).4. In Ringer's solution at 37°C the rates of K⁺ and Tl⁺ uptake were both inhibited by about 55% in ouabain and 60% in furosemide, bumetanide, or Na⁺-or Cl^–-free medium. This indicated that the routes for K⁺ and Tl⁺ uptake were similar and due to Na,K-ATPase-dependent transport and to Na-K-Cl cotransport.5. About 10% of the uptake was neither ouabain nor bumetanide sensitive. Ba²⁺, which is known to block inward-rectifying K⁺ channels and to depolarize glial cells, and other K⁺ channel blockers (Cs⁺ and bupivacaine), had no effect on Tl⁺ uptake.6. Metabolic inhibition with dinitrophenol reduced the uptake rate to 17%.7. The washout of Tl⁺ was unaffected by bumetanide and K⁺ channel blockers, but dinitrophenol caused a transient increase of 75%, an effect which persisted in the presence of K⁺ channel blockers.8. It was concluded that the high specific K⁺ and Tl⁺ accumulation in cultured human glioma cells was due not to the presence of inwardly rectifying K⁺ channels or other identified K⁺ channels, but to Na,K-ATPase dependent transport and Na-K-Cl cotransport.     

Fas antigen and bcl-2 expression on lymphocytes cultured with cytomegalovirus and varicella—zoster virus antigen

Fas antigen and bcl-2 expression on peripheral blood mononuclear cells (PBMC) cultured with cytomegalovirus (CMV) and varicella—zoster virus (VZV) antigen was analyzed by three-color flow cytometry. Expression of Fas antigen increased significantly in CD45RO⁺ populations of both CD4⁺ and CD8⁺ cells obtained from CMV and VZV seropositive donors after culture with CMV and VZV antigen for 6 days. Fas antigen expression did not increase in PBMC cultured with control antigen. In contrast, bcl-2 expression decreased both in CD4⁺CD45RO⁺ and CD8⁺CD45RO⁺ cells from the same donors. Fas antigen and bcl-2 expression in CD45RA⁺ populations did not change. Cell viability of cultured cells with CMV and VZV antigen decreased after treatment with anti-Fas antibody and DNA fragments indicating apoptosis were detected in the cell lysate of these cultured cells after treatment with anti-Fas antibody. These data suggest that Fas antigen and bcl-2 protein may interact in regulating the cell death process of activated memory lymphocytes and eliminating lymphocytes activated by viral infection.     

The Relationship between Cell Membrane Potassium Ion Transport and Glycolysis : The effect of ethacrynic acid

Cell membrane transport of K⁺ stimulates the rate of glycolysis in Ehrlich ascites tumor cells. A study of the characteristics of this relationship indicates that the stimulation occurs under anaerobic as well as under aerobic conditions. The data suggest that glycolysis is stimulated by a K⁺ transport mechanism that is coupled to Na⁺ transport because the effect is blunted or abolished when the principal intracellular ion is lithium or choline. This stimulus to glycolysis is blocked by ouabain and ethacrynic acid, agents that have been shown to inhibit monovalent cation transport in erythrocytes. In contrast to the action of ouabain, glycolysis is inhibited by ethacrynic acid in Ehrlich ascites tumor cells in the absence of cell membrane K⁺ transport. In studies with ghost-free hemolysates of human erythrocytes and with cytosol prepared from Ehrlich ascites tumor cells, ethacrynic acid significantly blocks lactate formation from fructose diphosphate demonstrating the direct inhibitory effect of this agent on one or more enzymes of the Embden-Meyerhof pathway. Since ethacrynic acid has no influence on lactate formation in intact erythrocytes utilizing an endogenous substrate, the presumptive site of inhibition is proximal to the 3-phosphoglycerate level.     

Leukocyte-associated immunoglobulin-like receptor-1 is expressed on human megakaryocytes and negatively regulates the maturation of primary megakaryocytic progenitors and cell line

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is an inhibitory collagen receptor which belongs to the immunoglobulin (Ig) superfamily. Although the inhibitory function of LAIR-1 has been extensively described in multiple leukocytes, its role in megakaryocyte (MK) has not been explored so far. Here, we show that LAIR-1 is expressed on human bone marrow CD34⁺CD41a⁺ and CD41a⁺CD42b⁺ cells. LAIR-1 is also detectable in a fraction of human cord blood CD34⁺ cell-derived MK that has morphological characteristics of immature MK. In megakaryoblastic cell line Dami, the membrane protein expression of LAIR-1 is up-regulated significantly when cells are treated with phorbol ester phorbol 12-myristate 13-acetate (PMA). Furthermore, cross-linking of LAIR-1 in Dami cells with its natural ligand or anti-LAIR-1 antibody leads to the inhibition of cell proliferation and PMA-promoted differentiation when examined by the MK lineage-specific markers (CD41a and CD42b) and polyploidization. In addition, we also observed that cross-linking of LAIR-1 results in decreased MK generation from primary human CD34⁺ cells cultured in a cytokines cocktail that contains TPO. These results suggest that LAIR-1 is a likely candidate for an early marker of MK differentiation, and provide initial evidence indicating that LAIR-1 serves as a negative regulator of megakaryocytopoiesis.     

Lanthanum-induced alterations of cellular electrolytes in Ehrlich ascites tumor cells: a new view

We have shown previously that Ehrlich ascites tumor cells maintained at room temperature under an oxygen atmosphere lose Na⁺, K⁺ and Cl^? isosmotically when exposed to La⁺⁺⁺ (0.1 to 1.0 mM). Concomitant with these changes there is an increase in the recorded membrane potential (increasing intracellular negativity). The present studies further characterize the effect of La⁺⁺⁺ on electrolyte distribution. Ehrlich ascites tumor cells were maintained at 0.5° C to permit Na⁺ gain and K⁺ loss. The addition of 1 mM La⁺⁺⁺ to low temperature cells induces rapid loss of Na⁺, K⁺ and Cl^?. This net loss of cellular electrolytes occurs even in cells depleted of ATP content using 2-deoxyglucose (5 mM) and rotenone (10^?6 M ). Analysis of the appearance of tracer ²²Na in the environment of cells preloaded with the radioisotope shows that La⁺⁺⁺-induced changes in membrane permeability or in active ion transport mechanisms are not responsible for the dramatic loss of electrolytes from experimental cells. The electrolyte loss occurs only when the cells are resuspended mechanically during the washing procedure used to prepare the cells for electrolyte determination. We conclude that the results of La⁺⁺⁺ interaction with Ehrlich ascites tumor cells are twofold. As we have previously reported, La⁺⁺⁺ stabilizes and causes a hyperpolarization of the membrane potential. Secondly, La⁺⁺⁺ predisposes the cell membrane to become highly permeable when subjected to mechanical stress.