Sodium influx in human leucocytes: A novel technique for assessment of sodium–proton antiport activity

1988 ◽  
Vol 75 (2) ◽  
pp. 179-184 ◽  
Author(s):  
L. L. Ng ◽  
M. Harker
Keyword(s):  
Ammonium Chloride ◽  
Intracellular Acidification ◽  
Maximal Inhibition ◽  
Sodium Influx ◽  
Novel Technique ◽  
Isotope Method ◽  
Uptake Rates ◽  
Normal Human ◽  
22Na Uptake ◽  
Na Depletion

1. A triple-isotope method for measuring initial 22Na uptake rates in unactivated human leucocytes in the presence of serum, using 3H2O, [14C]sucrose and 22Na, is described. 2. This 22Na influx is inhibited by amiloride, with half-maximal inhibition at a concentration of 10−5 mol/l. 3. About 70% of the 22Na influx in normal human leucocytes is inhibited by 1 mmol/l amiloride, with an external Na+ concentration of 10 mmol/l. 4. External Na+ antagonizes this inhibition by amiloride. The Km for external Na+ is 9 mmol/l. 5. Intracellular acidification from either external Na+ depletion or ammonium chloride incubation leads to an activation of amiloride-sensitive Na+ influx. 6. The amiloride-sensitive Na+–H+ antiport provides the major influx pathway for Na+ in unactivated human leucocytes in the presence of serum.

Mechanisms of leucocyte sodium influx in essential hypertension

1988 ◽  
Vol 75 (5) ◽  
pp. 521-526 ◽  
Author(s):  
L. L. Ng ◽  
M. Harker ◽  
E. D. Abel
Keyword(s):  
Essential Hypertension ◽  
Sodium Influx ◽  
Hypertensive Patients ◽  
Isotope Method ◽  
Uptake Rates ◽  
Normotensive Subjects ◽  
22Na Uptake

1. Leucocyte Na+ influx in media containing 10 mmol/l Na+ was studied directly using a triple-isotope method for measuring initial 22Na uptake rates in 20 normal and 18 untreated hypertensive subjects. The effects of 1 mmol/l amiloride (a Na+-H+-antiport inhibitor) and 0.1 mmol/l bumetanide (a Na+, K+, Cl−-symport inhibitor) were also examined. 2. The total, amiloride-sensitive and bumetanide-sensitive influx rates were raised in hypertensive compared with normotensive subjects [median (range): total 0.63 (0.25–1.82) vs 0.40 (0.09–0.65) mmol min−1 l−1, P < 0.002; amiloride-sensitive 0.43 (0.18–1.56) vs 0.26 (0.04–0.48) mmol min−1 l−1, P < 0.002; bumetanide-sensitive 0.12 (−0.03 to 0.83) vs 0.02 (−0.25 to 0.21) mmol min−1 l−1, P < 0.005]. 3. We conclude that hypertensive patients have a raised leucocyte total Na+ influx when measured in media containing 10 mmol/l Na+ and that this is contributed mainly by amiloride-sensitive and bumetanide-sensitive Na+ influx mechanisms.

A Novel Technique for Observing the Internal Ultrastructure of Human Chromosomes with Known Karyotype

2008 ◽  
Vol 14 (4) ◽  
pp. 357-361 ◽  
Author(s):  
Mohammad Ghazizadeh ◽  
Yoshihiro Sasaki ◽  
Tatsuo Oguro ◽  
Shigeru Sato ◽  
Seiko Egawa ◽  
...  
Keyword(s):  
Metaphase Chromosome ◽  
Osmium Tetroxide ◽  
Human Lymphocytes ◽  
Fragile Sites ◽  
Human Chromosomes ◽  
Chromosome Research ◽  
Novel Technique ◽  
Transmission Electron ◽  
Comprehensive Information ◽  
Normal Human

Observation of the internal ultrastructure of human chromosomes by transmission electron microscopy (TEM) has frequently been attempted in spite of the difficulties in detaching metaphase chromosome spreads from the glass slide for further processing. In this study we have used a method in which metaphase chromosome spreads were prepared on a flexible thermoplastic membrane (ACLAR) film. To assess chromosome identity, a diamidino-phenylindole staining and karyotying was first done using a conventional cytogenetic system. The chromosome spreads were then fixed with 1% osmium tetroxide, stained with freshly prepared 2% tannic acid, dehydrated, and flat-embedded in epoxy resin. The resin sheet was easily detachable and carried whole chromosome spreads. By this method, TEM observation of chromosomes from normal human lymphocytes allowed a thorough examination of the ultrastructure of centromeres, telomeres, fragile sites, and other chromosomal regions. Various ultrastructural patterns including thick electron dense boundaries, less dense internal regions, and extended chromatin loops at the periphery of the chromosomes were discernible. Application of the present method to chromosome research is expected to provide comprehensive information on the internal ultrastructure of different chromosomal regions in relation to function.

scholarly journals An immunotoxin with therapeutic potential in T cell leukemia: WT1-ricin A

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 1178-1185 ◽  
Author(s):  
CD Myers ◽  
PE Thorpe ◽  
WC Ross ◽  
AJ Cumber ◽  
FE Katz ◽  
...  
Keyword(s):  
T Cell ◽  
Ammonium Chloride ◽  
Therapeutic Potential ◽  
Autologous Transplantation ◽  
Lymphocytic Leukemia ◽  
Normal Human ◽  
A Chain ◽  
Density Treatment ◽  
Normal Human Bone Marrow ◽  
Ricin A

Abstract A conjugate of the monoclonal antibody WT1 and ricin A-chain was studied for its suitability for purging marrow of leukemic T cells for autologous transplantation in T cell acute lymphocytic leukemia (T- ALL). The conjugate was powerfully cytotoxic to the human T-ALL cell line, GH1, which expresses the WT1 antigen at a high density. Treatment of the cells with the conjugate at 10(-11) M reduced their rate of protein synthesis by 50%, and the inclusion of 6 mM ammonium chloride in the cultures enhanced the potency of cytotoxic effect by 10–100- fold. Clonogenic assays indicated that less than 0.1% of GH1 cells survived 3-hr exposure to the conjugate in ammonium chloride. WT1 alone did not react with multipotent (CFU-GEMM) hematopoietic progenitors in normal human bone marrow, as measured by fluorescence-activated cell sorting. Under conditions giving maximal killing of GH1 cells, there was no toxicity to multipotential progenitors in normal human marrow.

Frusemide-Sensitive Sodium and Potassium Transport by Human Leucocytes

1985 ◽  
Vol 68 (1) ◽  
pp. 89-91 ◽  
Author(s):  
Valerie E. Johnson ◽  
P. J. Hilton
Keyword(s):  
Potassium Transport ◽  
Sodium Influx ◽  
Potassium Efflux ◽  
Sodium Potassium ◽  
External Potassium ◽  
Normal Human ◽  
Potassium Influx ◽  
Net Flux ◽  
Sodium And Potassium

1. Frusemide-sensitive sodium and potassium transport by normal human leucocytes has been studied in vitro by both isotopic and net flux techniques. 2. In physiological media the leucocyte exhibits a frusemide-sensitive influx of sodium and potassium of equal magnitude compatible with a 1:1 co-transport system. 3. Cells exposed to zero external sodium and potassium (osmolality maintained with choline) demonstrated a frusemide-sensitive sodium and potassium efflux. 4. Frusemide-sensitive potassium influx was dependent on the presence of external sodium but frusemide-sensitive sodium influx persisted unchanged in the absence of external potassium. 5. Frusemide-sensitive potassium influx was dependent on external chloride but frusemide-sensitive sodium influx was chloride-independent. 6. These last two observations make it likely that the frusemide-sensitive pathway is capable of operating in modes other than sodium-potassium co-transport.

ACTIVATION AND PHYSIOLOGICAL ROLE OF Na+/H+ EXCHANGE IN LAMPREY (LAMPETRA FLUVIATILIS) ERYTHROCYTES

1994 ◽  
Vol 191 (1) ◽  
pp. 89-105 ◽  
Author(s):  
L Virkki ◽  
M Nikinmaa
Keyword(s):  
Sodium Transport ◽  
Physiological Role ◽  
Cell Swelling ◽  
Intracellular Acidification ◽  
Adrenergic Stimulation ◽  
Sodium Influx ◽  
Lampetra Fluviatilis ◽  
Physiological Conditions ◽  
Red Cell Ph

The effects of intracellular acidification, osmotic shrinkage and ss-adrenergic stimulation on sodium transport across the membrane of lamprey (Lampetra fluviatilis) erythrocytes were investigated. Unidirectional ouabain-insensitive sodium flux, measured using radioactive 22Na, was increased markedly by intracellular acidification, to a lesser extent by osmotic shrinkage and only modestly by ss-adrenergic stimulation. Na+/H+ exchange was activated in all of these cases. However, net sodium influx (and cell swelling caused by the influx of osmotically obliged water) was seen only in cells subjected to intracellular acidification. In contrast, practically no changes in red cell pH or in water or ion (Na+, K+ and Cl-) contents were seen after osmotic shrinkage or ss-adrenergic stimulation. Calculations of the [Na+]o/[Na+]i and [H+]o/[H+]i ratios across the erythrocyte membrane suggest that the virtual lack of net sodium movements in osmotically shrunken erythrocytes is due to the absence of a driving force for net transport of these ions via the Na+/H+ exchange pathway. It also appears that, in physiological conditions, the increase in the activity of the Na+/H+ exchanger by ss-adrenergic stimulation is too small to mediate detectable net sodium transport.

Transport of n-butyrate into human colonic luminal membrane vesicles

1996 ◽  
Vol 271 (3) ◽  
pp. G415-G422 ◽  
Author(s):  
J. M. Harig ◽  
E. K. Ng ◽  
P. K. Dudeja ◽  
T. A. Brasitus ◽  
K. Ramaswamy
Keyword(s):  
Short Chain Fatty Acid ◽  
Membrane Vesicles ◽  
Organ Donor ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
Luminal Membrane ◽  
Uptake Rates ◽  
In Trans ◽  
22Na Uptake ◽  
Stimulation Of

Human colonic short-chain fatty acid (SCFA) absorption is associated with increased luminal pH and HCO3- and enhanced Na+ absorption. Therefore, the mechanism of colonic SCFA transport, its dependence on Na+ and HCO3- and interactions with Cl-/HCO3- and Na+/H+ exchangers were characterized. Luminal membrane vesicles (LMV) isolated by divalent cation precipitation from organ donor colons were used for n-butyrate transport. Uptake of n-butyrate into the human colonic LMV was minimal even in the presence of an inward pH gradient, but an outward HCO3- gradient significantly increased uptake rates. HCO3(-)-stimulated butyrate uptake was saturable with an apparent Michaelis constant of 1.5 +/- 0.2 mM and maximal velocity of 105 +/- 3 nmol.mg protein-1.3 s-1. Intravesicular butyrate resulted in trans-stimulation of n-[1-14C]butyrate uptake. Butyrate uptake was inhibited approximately 25-40% by C2-C5 SCFAs and approximately 40% by niflumic acid. Butyrate uptake was not affected by extravesicular Na+, and 22Na uptake was unaltered by extravesicular butyrate. Butyrate uptake was independent of extra- or intravesicular CI-, and butyrate loading produced no changes in 36Cl uptake. We conclude that the predominant mechanism of n-butyrate transport across the human colonic luminal membrane appears to be via a HCO3-/SCFA antiport system independent of Cl-/HCO3- exchange and Na+ transport.

Inhibition of normal human granulopoiesis in vitro by amiloride: Evidence for the involvement of facilitated sodium influx in the induction of differentiation

1987 ◽  
Vol 11 (8) ◽  
pp. 719-724 ◽  
Author(s):  
Andrew J. Steed ◽  
Roman Pylypczuk ◽  
Raymond C. Routledge ◽  
Irvine W. Delamore
Keyword(s):  
Sodium Influx ◽  
Normal Human ◽  
Induction Of Differentiation

Na+-H+ exchange activity in rat hepatocytes: role in regulation of intracellular pH

1989 ◽  
Vol 256 (1) ◽  
pp. G44-G52 ◽  
Author(s):  
E. L. Renner ◽  
J. R. Lake ◽  
M. Persico ◽  
B. F. Scharschmidt
Keyword(s):  
Intracellular Ph ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Rat Hepatocytes ◽  
Rate Of Change ◽  
Intracellular Acidification ◽  
22Na Uptake ◽  
Approach Zero ◽  
Na Uptake ◽  
Exchange Activity

Amiloride-sensitive Na+-H+ exchange has been identified in basolateral membrane vesicles from rat liver, but little is currently known about its regulation or its role in maintenance of resting intracellular pH (pHi) in intact hepatocytes. We have assessed Na+-H+ exchange activity in isolated or cultured rat hepatocytes in nominally HCO3- free solution under basal conditions and after intracellular acidification by an NH4Cl pulse by measuring 1) pHi, using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxy fluorescein, 2) net H+ efflux by pH-stat titration, and 3) amiloride-inhibitable 22Na uptake. Under resting conditions, Na+-H+ exchange did not contribute measurably to Na+ uptake and accounted for less than 20% of net H+ efflux. Hepatocyte pHi averaged 7.07 +/- 0.03, significantly above H+ electrochemical equilibrium (6.92 +/- 0.08) determined using an electrogenic proton ionophore. Transient removal of extracellular Na+ or exposure to amiloride reversibly lowered pHi by 0.09 +/- 0.01 and 0.12 +/- 0.03 pH units, respectively, within 5-10 min. After intracellular acidification by an NH4Cl pulse, Na+ uptake rate increased about twofold, the increase being entirely amiloride inhibitable. Net H+ efflux increased about threefold, and 70% of the increase was amiloride inhibitable. Recovery of pHi after an NH4Cl pulse was reversibly blocked by exposure to amiloride or removal of Na+. Na+-H+ exchange activity (calculated from the rate of change in pHi and intracellular buffering capacity) was inversely related to pHi and was estimated to approach zero at pHi 7.25-7.50.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium and Potassium Transport Rates in Normal Human Leucocytes in Hypo-Osmolal Extracellular Fluid

1974 ◽  
Vol 46 (5) ◽  
pp. 613-617 ◽  
Author(s):  
P. J. Hilton ◽  
J. Patrick
Keyword(s):  
Rate Constants ◽  
Extracellular Fluid ◽  
Dry Weight ◽  
Potassium Transport ◽  
Sodium Influx ◽  
Potassium Efflux ◽  
Normal Human ◽  
Potassium Influx ◽  
Sodium And Potassium

1. Sodium and potassium transport rates were studied in normal human leucocytes exposed to iso-osmolal and hypo-osmolal extracellular fluid. 2. Hypo-osmolality of the extracellular fluid led to an increase in sodium influx and a decrease in potassium influx expressed as mmol h−1 kg−1 cell dry weight. The fall in potassium influx was smaller than the rise in sodium influx and was confined to the ouabain-insensitive portion of the flux. 3. The rate constants for sodium and potassium efflux did not differ significantly between the iso-osmolal and hypo-osmolal media.

scholarly journals An immunotoxin with therapeutic potential in T cell leukemia: WT1-ricin A

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 1178-1185
Author(s):  
CD Myers ◽  
PE Thorpe ◽  
WC Ross ◽  
AJ Cumber ◽  
FE Katz ◽  
...  
Keyword(s):  
T Cell ◽  
Ammonium Chloride ◽  
Therapeutic Potential ◽  
Autologous Transplantation ◽  
Lymphocytic Leukemia ◽  
Normal Human ◽  
A Chain ◽  
Density Treatment ◽  
Normal Human Bone Marrow ◽  
Ricin A

A conjugate of the monoclonal antibody WT1 and ricin A-chain was studied for its suitability for purging marrow of leukemic T cells for autologous transplantation in T cell acute lymphocytic leukemia (T- ALL). The conjugate was powerfully cytotoxic to the human T-ALL cell line, GH1, which expresses the WT1 antigen at a high density. Treatment of the cells with the conjugate at 10(-11) M reduced their rate of protein synthesis by 50%, and the inclusion of 6 mM ammonium chloride in the cultures enhanced the potency of cytotoxic effect by 10–100- fold. Clonogenic assays indicated that less than 0.1% of GH1 cells survived 3-hr exposure to the conjugate in ammonium chloride. WT1 alone did not react with multipotent (CFU-GEMM) hematopoietic progenitors in normal human bone marrow, as measured by fluorescence-activated cell sorting. Under conditions giving maximal killing of GH1 cells, there was no toxicity to multipotential progenitors in normal human marrow.

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