Bretylium-induced voltage-gated sodium current in human lymphocytes.

Using the whole-cell variation of the patch-clamp technique it has been determined that 0.25-3 mM bretylium tosylate (BT) exerts a repolarizing effect on partially depolarized human lymphocytes. The repolarizing effect was ouabain (40 microM)-sensitive, and was inhibited by the removal of external Na+ or by the Na(+)-channel-blocker amiloride (10-44 microM), but K(+)-channel-blockers 4-aminopyridine (0.1-5 mM) and quinine (100 microM) had no effect. The drug induced a sodium dependent, amiloride-sensitive transient inward current reaching its maximum value approx. 20-30 s after the administration of BT and lasting for 6-10 min. This current was activated by depolarization within 25 ms at around -42 mV, its inactivation took about 2 s and its reversal potential was +24 +/- 5 mV. An increase in the intracellular sodium concentration (1.8-3.2 mM) has been observed upon the addition of BT by monitoring the SBFI fluorescence of the dye-loaded cells. It has been shown that whole-cell K+ currents are significantly decreased by BT. The existence of voltage and ligand (BT)-gated sodium channels has been postulated in human lymphocytes. These channels are thought to participate in the initiation of membrane repolarization in human lymphocytes, and thereby influence mitogenic or antigen-induced cell-activation processes.     

Cytogenetic damage induced in human lymphocytes by sodium bisulfite.

The frequencies of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), and micronuclei (MN) in human blood lymphocytes exposed to sodium bisulfite (sulfur dioxide) at various concentrations ranging from 5 x 10(-5) M to 2 x 10(-3) M in vitro were studied. It was shown that sodium bisulfite (NaHSO3 and Na2SO3, 1:3 M/M) caused an increase in SCE and MN in human blood lymphocytes in a dose-dependent manner, and also induced mitotic delays and decreased mitotic index. For CA, our results indicated that sodium bisulfite induced an increase of chromatid-type aberrations in lymphocytes from three of four donors in a dose-dependent manner. The chemical at low concentrations induced chromatid-type aberrations, but not chromosome-type aberrations; high concentrations induced both chromatid- and chromosome-type aberrations. No cytogenetic damage in human lymphocytes was induced by sodium sulfate. The results have confirmed that sulfur dioxide is a clastogenic and genotoxic agent.     

Multiple fractions of sodium exchange in human lymphocytes

Human lymphocytes contain a large, saturable fraction of K⁺ that exchanges slowly with K⁺ in the external medium, and a small non-saturable fraction that exchanges rapidly. We determined whether or not Na⁺ exchanges in a similar manner with external Na⁺. Cells were pre-equilibrated to ensure absence of net ion movements. Efflux was studied by loading with ²²Na and transferring without washing to a non-labeled medium. Influx was studied by transferring to labeled medium and separating large samples of cells at 6,000g. There are fast, intermediate, and slow fractions of Na⁺ exchange, with half-times of 2, 14, and 120 minutes. At normal external K⁺, most cell Na⁺ exchanges rapidly, while at lower external K⁺ the Na⁺ that replaces cell K⁺ exchanges slowly. Parallel sources of fast and slow fractions, such as extracellular ones and subpopulations of cells, were ruled out by simultaneous ⁴²K and ²²Na fluxes and by a quantitative analysis of the combined K⁺ and Na⁺ content and flux data over a range of external K⁺ and Na⁺ levels. Five possible models of ion fluxes occurring in series were considered. Surface matrix, surface binding sites, and cytoplasmic channels with rapid nuclea exchange were eliminated as sources of the fast fractions. Therefore, the fast fractions of K⁺ and Na⁺ must reflect the permeability of the surface membrane. This left only two possible sources of the slow fractions. One, a subcellular compartment (e.g., nucleus), was eliminated by the combined content and flux data. We conclude that the slow fractions of ion flux are rate-limited by adsorption onto and desorption from cellular macromolecules. The data support the association-induction hypothesis and are understood by reference to two fundamental concepts: that of rapid solute exclusion from cell water existing in a polarized state; and that of solute accumulation limited by adsorption onto fixed anionic sites within the cell.     

Genotoxicity of food preservative sodium sorbate in human lymphocytes in vitro

The genotoxic effects of antimicrobial food additive sodium sorbate (SS) was assessed by using chromosome aberrations (CAs), sister-chromatid exchanges (SCEs), and micronucleus (MN) in cultured human lymphocytes and comet assay in isolated human lymphocytes. Lymphocytes were treated with four concentrations (100, 200, 400 and 800 μg/ml) of SS as well as a negative (sterile distilled water) and a positive control (Mitomycin-C: MMC for cultured lymphocytes and H₂O₂ for isolated lymphocytes). The result of this study indicated that SS increased the frequency of CAs at both 24 and 48 h period compared to control. When gaps were included, this increase was significant at 200, 400 and 800 μg/ml concentrations at 24 h and, at all concentrations at 48 h treatment time. When gaps were excluded, this increase was significant at only 800 μg/ml concentration at both 24 and 48 h treatments. In addition, SS increased SCEs/cell and MN frequency at 400 and 800 μg/ml concentrations at both 24 and 48 h compared to negative control. Furthermore, this additive caused DNA damage at all concentrations in isolated human lymphocytes after 1 h in vitro exposure. The present results show that SS is genotoxic to the human peripheral blood lymphocytes in vitro at the highest concentrations.     

Extracellular ATP stimulates an amiloride-sensitive sodium influx in human lymphocytes

Extracellular ATP has been shown to increase the Na+ permeability of human lymphocytes by 3 to 12-fold. The kinetics of this ATP-induced response were studied by measuring 22Na+ influx into chronic lymphocytic leukemic lymphocytes incubated in low-sodium media without divalent cations. ATP-stimulated uptake of 22Na-ions was linear over 4 min incubation and this influx component showed a sigmoid dependence on ATP concentration. Hill analysis yielded a K1/2 of 160 microM and a n value of 2.5. The nucleotide ATP-gamma-S (1-2 mM) gave 30% of the permeability increase produced by ATP, but UTP (2 mM) and dTTP (2 mM) had no effect on 22Na influx. The amiloride analogs 5-(N-ethyl-N-isopropyl) amiloride and 5-(N,N-hexamethylene) amiloride, which are potent inhibitors of Na(+)-H+ countertransport, abolished 72-95% of the ATP-stimulated 22Na+ influx. However, the involvement of Na(+)-H+ countertransport in the ATP-stimulated Na+ influx was excluded by three lines of evidence. Sodium influx was stimulated 7-fold by extracellular ATP but only 2.4-fold by hypertonic conditions which are known to activate Na(+)-H+ countertransport. Addition of ATP to lymphocytes produced no change in intracellular pH when these cells were suspended in isotonic NaCl media. Finally ATP caused a membrane depolarization of lymphocytes which is inconsistent with stimulation of electroneutral Na(+)-H+ exchange. These data suggest that ATP acts cooperatively to induce the formation of membrane channels which allow increased Na+ influx by a pathway which is partially inhibited by amiloride and its analogs.     

Effects of ascorbic acid and sodium ascorbate on cyclic nucleotide metabolism in human lymphocytes.

L-ascorbic acid (LAA) augmented cGMP many-fold in highly purified human peripheral blood lymphocytes. The cGMP response occurred within 10 sec and persisted for at least 60 min. D-ascorbic acid (DAA) and dehydroascorbic acid (DHAA) were also equally active in enhancing cGMP concentrations but metabolic precursors of ascorbic acid and other inorganic acids did not increase cGMP levels. Determination of the amount of DHAA contaminating the LAA precluded the possibility that it was solely responsible for the enhanced cGMP levels. The sodium or calcium salts of ascorbic acid did not increase cGMP concentrations. If these neutralized preparations were acidified, increased cGMP concentrations were then noted. In broken cell preparations, LAA, DAA, and DHAA and to a lesser extent sodium ascorbate (NaA) enhanced guanylate cyclase activity while neither inhibited cAMP or cGMP phosphodiesterase (PDE) activity. The possible role of H2O2, fatty acid liberation, prostaglandin production, oxidizing-reducing agents, and free radical formation in mediating the effects of ascorbic acid on cGMP levels were evaluated, but none of these potential mechanisms were definitively proven to be a required intermediary for the cGMP enhancing activity of ascorbic acid. LAA, DHAA or NaA did not induce lymphocyte transformation or modulate lectin-induced mitogenesis.     

A sodium requirement for mitogen-induced proliferation in human peripheral blood lymphocytes

Mitogen-stimulated DNA synthesis in human peripheral blood lymphocytes is dependent on extracellular Na. DNA synthesis was similarly inhibited in:

1. 1. Cells that were suspended in hypotonic media containing decreased extracellular Na.

2. 2. Cells that were suspended in media containing decreased Na and equimolar replacement with choline.

3. 3. Cells that were suspended in media containing decreased Na and equiosmolar replacement with mannitol.

A decreased PHA-induced DNA synthesis was observed at day 3 even when lymphocytes were exposed to low Na for only the first 3 h and then returned to normal levels of Na. Our studies of protein synthesis indicate that the effect of lowered extracellular Na on DNA synthesis and cell division is not due to an initial inhibition of overall protein synthesis. These data suggest that reduced external Na has a significant effect on some specific early event(s) (3 h) in lymphocyte mitogenesis.     

Effects of sodium butyrate on X-ray and bleomycin-induced chromosome aberrations in human peripheral blood lymphocytes.

Peripheral blood lymphocytes from normal human volunteers or from Down syndrome patients were pre-treated with sodium butyrate (a compound which is known to induce structural modifications in the chromatin through hyperacetylation of nucleosomal core histones) and exposed to X-irradiation or treated with bleomycin in vitro in the G0 and/or G1 stage(s) of the cell cycle. The frequencies of chromosomal aberrations in the first mitosis after treatment were scored. The results show an enhancement in the yield of aberrations in the butyrate pre-treated groups. However, the absolute frequencies of chromosomal aberrations as well as the relative increases with butyrate pre-treatment varied between blood samples from different donors suggesting the existence of inter-individual variations. There is a parallelism between the effects of X-irradiation or of combined treatments in G0 and G1 stages and between effects observed in the X-ray and bleomycin series. The increase in the yields of chromosomal aberrations in butyrate-treated and X-irradiated lymphocytes (relative to those which received X-irradiation alone) is interpreted as a consequence of the inhibition of repair of DNA damage by butyrate.     

Purine metabolism in human lymphocytes.

In peripheral human blood lymphocytes the uptake and metabolism of adenine, guanine, and hypoxanthine was investigated. This was achieved by incubation of purified lymphocytes with 14C-purine bases, separation of cells from the incubation medium by a rapid filtration technique, and subsequent separation of the acid soluble material by thin-layer chromatography. No perferential uptake for one of the purine bases was observed. In all cases only traces of 14C-purine bases not added originally and labeled nucleosides could be demonstrated. Approximately 2/3 of adenine and 1/2 of guanine or hypoxanthine were converted to nucleotides. Separation of formed nucleotides showed that adenine and guanine were metabolized mainly to their corresponding nucleotides; hypoxanthine was converted to a considerable amount to adenine nucleotides and only to a small proportion into its own nucleotides. These results demonstrate the predomonance of adenine nucleotide formation in normal human lymphocytes.     

Cytotoxic and genotoxic effects of sodium hypochlorite on human peripheral lymphocytes in vitro

Chlorination is widely used method in the disinfection of drinking and utility water worldwide. In this study, cytotoxic and genotoxic effects of sodium hypochlorite were investigated by the cytokinesis-block micronucleus assay and chromosomal aberration analysis on human peripheral lymphocytes in vitro. A significant increase in chromosomal aberration frequency was observed in all treatments of NaOCl (0.030, 0.065, 0.100, 0.25, 0.5, 1, 2, 4 μg/mL) at 24 and 48 h compared with the negative control and mitomycin C (MMC, 0.3 μg/mL), which was used as a positive control. NaOCl significantly increased the frequency of micronuclei in a dose dependent manner. The results showed that there was a significant correlation between NaOCl concentration and chromosomal aberration, micronuclei frequency, necrotic cells, apoptotic cells and binucleated cells.     

Micronucleus assay in human lymphocytes after exposure to alloxydim sodium herbicide in vitro

This study evaluates the cytotoxic and genotoxic potential of alloxydim sodium using micronucleus (MN) assay, in human peripheral lymphocytes. MN assay was used to investigate the genotoxic effects of alloxydim sodium in human peripheral lymphocytes treated with 250, 500, 750, 1,000 µg/ml concentrations of alloxydim sodium for 24 and 48 h. Solvent, negative and positive controls were also used in the experiments in parallel. The obtained results were evaluated in statistical analyses by using Dunnett-t test (two sided) and p < 0.05 was accepted as significant. Alloxydim sodium significantly increased the MN formation compared with the negative control, at both 750 and 1,000 µg/ml concentrations and treatment periods. We also evaluated the nuclear division index (NDI) for cytotoxicity of this pesticide in the experiment, and finally observed a significant decrease of the NDI values at all concentrations of alloxydim sodium and at both treatment periods.     

Surface alterations in calf lymphocytes oxidized by sodium periodate.

In order to investigate alterations in surface structure in transformed lymphocytes, calf submandibular lymph node cell suspensions were oxidized with NaIO4. Oxidezed lymphocytes were morphologically transformed and had higher rates of DNA synthesis by 2 days after treatment. These results were prevented by reduction of the cell suspension with NaBH4, or by neuraminidase treatment of cells prior to oxidation. The amount of 125I-labeled Agaricus bisporus lectin bound to cells immediately after oxidation and the affinity constant for binding were increased over 2-fold, while cells immediately following oxidation and reduction showed decreased receptors with still higher affinity for the lectin compared to untreated cells. The amount of Phaseolus vulgaris lectin bound to oxidezed cells was also increased, but affinity was unchanged. Immediately following oxidation and reduction, these receptor sites were unchanged in number and affinity from untreated cells. In contrast, the number and affinity of receptors for concanavalin A were not changed immediately after oxidation or oxidation and reduction. In order to define the extent of compositional changes in surface glycoprotein receptors, plasma membranes were isolated from frozen calf submandibular lymph nodes. Compared to untreated plasma membranes, oxidezed membranes had similar contents of galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, fucose, and amino acids. Sialic acid content of oxidized membranes was reduced when measured by thiobarbituric acid assay. Sialic acids of untreated plasma membranes co-chromatographed with N-glycolylneurominic acid and N-acetylneuraminic acid, while those of oxidized membranes co-chromatographed with N-glycolylneuraminic acid and 5-acetamido-3,5-dideoxy-L-arabino-7-aldehydo-2-heptulosonic acid. Therefore, specific surface conformational changes in certain classes of membrane glycoproteins are associated with mild Malapradian oxidation of membrane sialic acids. These temporally precede NaIO4-induced transformation of calf lymphocytes. This is consistent with an hypothesis of membrane-mediated stimulation of lymphocyte transformation.     

D609-phosphatidylcholine-specific phospholipase C inhibitor attenuates thapsigargin-induced sodium influx in human lymphocytes

Previously, we reported that the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor tricyclodecan-9-yl xanthogenate (D609) potentiates thapsigargin-induced Ca(2+) influx in human lymphocytes. In the present study we examined the effect of D609 on the thapsigargin-induced Na(+) entry. We found that the early phase of the thapsigargin-induced increase in the intracellular Na(+) concentration (approx. 1-2 min after stimulation) was attenuated after preincubation of lymphocytes with D609. By contrast, thapsigargin-induced Na(+) influx was not affected in the presence butan-1-ol, which inhibits phosphatidylcholine-specific phospholipase D (PC-PLD). The thapsigargin-induced Na(+) influx could be mimicked by PC-PLC exogenously added to the lymphocyte suspension, whereas addition of PC-PLD had no effect. In addition, thapsigargin stimulated formation of the physiological PC-PLC products, diacylglycerol. Cell-permeable diacylglycerol analogue, dioctanoyl-glycerol (DOG), produced time- and concentration-dependent increase in the intracellular Na(+) concentration. Both thapsigargin- and DOG-induced Na(+) increases were not affected in the presence of Na(+)/H(+) antiport inhibitor, HOE609, or Na(+)/Ca(2+) antiport inhibitor, dimethylthiourea, as well as in the presence of Co(2+) and Ni(2+), which block store-operated Ca(2+) entry. By contrast, markedly reduced thapsigargin- and DOG-induced Na(+) influx were noted in the presence of flufenamic acid, which blocks the non-selective cation current (I(CRANC)). In conclusion, our results suggest that diacylglycerol released due to the PC-PLC activation contributes to the thapsigargin-induced Na(+) entry.     

The effect of metabolic inhibition on ion contents and sodium exchange in human lymphocytes

Lymphocytes depleted of ATP by incubation in iodoacetate (IAA) and nitrogen (N2) lost K and gained Na. Isotopic Na exchange showed a fast fraction and a slower exponential fraction, the latter conventionally assumed to reflect surface membrane properties. The gain of cell Na was not accounted for by a decrease in 22Na efflux in either the slow or the fast fraction. After 3-5 hours, Na efflux increased. These results led us to question the concept that normal cell ion levels are maintained by an ATPase pump and could not be explained by exchange diffusion, co-transport, countertransport, or other inherently dissipative mechanisms. The data are, on the other hand, consistent with the concept that cell ion contents are determined by their relative exclusion from cell water coupled with selective adsorption onto fixed macromolecular anionic sites within the cell. In this view, the IAA,N2-induced rise in cell Na is due to the occupancy of adsorption sites losing K, while the increased isotopic exchange is due to a decreased activation energy for ion-site interaction.     

Glycosphingolipids of purified human lymphocytes.

Biochemical analysis of the glycosphingolipids (GSLs) of human lymphocytes revealed qualitative and quantitative variations among purified lymphocytes from different tissues. The major neutral GSLs of tonsil lymphocytes are glucosyl ceramide (CMH), lactosyl ceramide (CDH), trihexosyl ceramide (CTH), and globoside. Thymocytes and peripheral blood lymphocytes (PBL) contain only traces of CTH and globoside, and PBL contain more CMH and CDH per cell than tonsil lymphocytes. Thymocytes and PBL contain relatively large amounts of more complex neutral GSLs that are present in only trace amounts in tonsil lymphocytes. Peripheral blood lymphocytes contained three and five times more lipid-bound sialic acid than thymocytes and toncil lymphocytes, respectively. Thymocytes and PBL contained mostly hematoside, whereas tonsil lymphocytes contained more complex gangliosides in addition to hematoside. The observed differences in GSL content among these cells may be related to their content of B cells, which comprise approximately 50% of tonsil lymphocytes, 10% of PBL and 0-2% of thymus cells, and/or the known differences in functional capacities of cells in different lymphoid organs. These findings suggest that cell surface GSLs may serve as markers for identification of functional subpopulations of human lymphocytes.     

Phospholipase A(2) is involved in thapsigargin-induced sodium influx in human lymphocytes

Previously, we reported that emptying of intracellular Ca(2+) pools with endoplasmatic Ca(2+)-ATP-ase inhibitor thapsigargin leads to the Na(+) influx in human lymphocytes (M. Tepel et al., 1994, J. Biol. Chem. 269, 26239-26242). In the present study we examined the mechanism underlying the thapsigargin-induced Na(+) entry. We found that the thapsigargin-induced increase in Na(+) concentration was effectively inhibited by three structurally unrelated phospholipase A(2) (PLA(2)) inhibitors, p-bromophenacyl bromide, 3-(4-octadecyl)-benzoylacrylic acid (OBAA), and bromoenol lactone (BEL). The thapsigargin-induced Na(+) influx could be mimicked by PLA(2) exogenously added to the lymphocyte suspension. In addition, thapsigargin stimulated formation of arachidonic acid (AA), the physiological PLA(2) product. AA induced Na(+) entry in a time- and concentration-dependent fashion. Both, thapsigargin-induced Na(+) influx and AA liberation were completely inhibited in the presence of tyrosine kinase inhibitor genistein but not in the absence of extracellular Ca(2+). Collectively, these data show that thapsigargin-induced Na(+) entry is associated with tyrosine kinase-dependent stimulation of PLA(2).     

Effect of sodium azide on sister-chromatid exchanges in human lymphocytes and Chinese hamster cells

Previous reports from this laboratory and others indicate that sodium azide is a unique mutagen. It is highly mutagenic in S. typhimurium TA1530 as well as in barley, rice, peas, yeast and Chinese hamster V79 cells. However, azide apparently does not produce chromosome breaks in barley, Vicia or human lymphocytes. Therefore, a study of the effects of azide on sister-chromatid exchanges (SCE) appeared warranted.Human whole blood and Chinese hamster K1 cell line were exposed for 4 and 2 h resp. to various concentrations of sodium azide ranging from 10⁻³ to 10⁻⁷ M. Cells were harvested and chromosomes stained by the FPG technique. In human lumphocytes, concentrations above 10⁻⁴ induced lethality whereas the K1 cell line was sensitive to concentrations above 10⁻⁵ M. The lower concentrations of azide produced no significant increase in SCE frequency above controls. Concurrent mitomycin C treatments produced significant increases in SCE levels.This apparent lack of induction of SCEs above background combined with previous data demonstrating negative clastogenic but very positive mutagenic activity of azide confirms the uniqueness of this mutagen. It would appear that azide is one of the few known potent mutagens that does not increase SCEs and/or break chromosomes.     

Alkaline phosphatase in human lymphocytes. I. Cytochemical detection of alkaline phosphatase in normal human peripheral blood lymphocytes

The present paper deals with a sensitive cytochemical method of identifying alkaline phosphatase (AP) in rosette-forming lymphocytes gained from the peripheral blood of healthy human beings. The percentage of AP-positive lymphocytes amounts to 5%, with all cells comprising B- and O-lymphocyte population and with T-lymphocytes being negative. In a group of healthy test persons, recently, however, having undergone various inflammatory processes or virus diseases, the number of AP-positive lymphocytes is significantly higher, from 41-73% in B- and O-lymphocytes and from 6-38% in T-lymphocytes. This observation indicates that AP in lymphocytes may have a clinical significance in reactive lymphoproliferative processes, which must be elucidated by further investigations.