Ageing of rabbit red cells in vitro: membrane modifications and their possible role in red cell survival in vivo

In vitro incubation induces, in rabbit red cell membranes, significant modifications consisting mainly in a decrease of sialic acid and galactose. In vivo the life span of incubated erythrocytes seems to be correlated to the degree of surface alterations and ATP depletion: larger surface modifications and energy charge reduction induce shorter survival time. It can therefore be postulated that incubation of red cell in vitro can cause an ageing process similar to that occurring physiologically in vivo.     

Role of endonuclease activity and DNA fragmentation in Ca2+ ionophore A23187-mediated injury to rabbit isolated gastric mucosal cells

In the current study, the role of endonuclease activity in calcium ionophore A23187-induced gastric mucosal cellular disruption was examined using rabbit gastric mucosal cells. Cell integrity was assessed using trypan blue dye exclusion and Alamar blue dye absorbance. Ionophore A23187 (1.6–25 µM) induced a concentration-dependent decrease in dye exclusion and cell metabolism in cells suspended in a medium containing Ca²⁺ (2 mM), while no such effect was observed in cells incubated in the absence of extracellular Ca²⁺. Cells that were pretreated with the endonuclease inhibitors aurintricarboxylic acid (ATCA; 0.2 or 0.5 mM or Zn²⁺; 0.01 and 0.1 mM) exhibited significant reduction in the total extent of cell injury when incubated with A23187 in the presence of Ca²⁺. DNA fragmentation as assessed by measurement of [³H]thymidine liberation or gel electrophoresis was increased in response to ionophore A23187 (12.5 or 25 µM) treatment. A minimal degree of fragmentation was observed when cells were suspended in a Ca²⁺-free medium or incubated in the presence of ATCA or Zn²⁺. Addition of ethanol (8% w/v) induced a significant increase in cell injury, which was not affected by either removal of extracellular Ca²⁺ or ATCA pretreatment. Furthermore, treatment with the antioxidants catalase (50 µg/ml) or 2,2-dipyridyl (2 mM) reduced ionophore-induced cell injury but did not reduce the extent of DNA fragmentation. These data suggest that sustained increases in intracellular Ca²⁺ result in increased endonuclease activity in gastric mucosal cells, leading to extensive DNA lysis and cell damage. Ethanol-induced cell damage does not involve Ca²⁺ influx and therefore is not mediated by endonuclease activation. Furthermore, sustained increases in cellular Ca²⁺ may also mediate their effects via formation of reactive oxygen metabolites, but this mechanism of cell damage does not appear to involve DNA fragmentation.This work was supported by a grant from the Medical Research Council of Canada (MT 6426).     

Selective increase of potassium permeability in red blood cells exposed to acetylphenylhydrazine

Normal human red blood cells, when exposed briefly to acetylphenylhydrazine (APH), acquire Heinz bodies and a propensity for net ion and water loss upon subsequent incubation in an APH-free medium of physiologic sodium and potassium (K) content. The cells can be protected from APH damage by previous deoxygenation. The ion and water loss depend on the presence of a K gradient from cell to medium. In contradistinction to some other types of membrane perturbation in which K permeability is increased, the APH effect is not dependent on calcium. The meaning of these observations is discussed in relation to the vulnerability of the K permeability barrier.     

Altered response of stored red cells to Ca2+ stress

Short-term Ca2+ loading of erythrocytes was used as a test for probing membrane protein susceptibilities toward intrinsic enzymes in cells from fresh and from stored blood. The proteolytic response, affecting mainly glycophorin and band 3, could be elicited only in fresh cells, whereas the transglutaminase-mediated cross-linking reaction was evident both in fresh and stored cells. Loss of the proteolytic response might be an important sign of erythrocyte damage from blood bank storage.     

Production of arachidonic acid metabolites by macrophages exposed in vitro to asbestos, carbonyl iron particles, or calcium ionophore

Consequent to asbestos deposition, alveolar macrophages (AM) accumulate at alveolar duct bifurcations where they phagocytize fibers. Because phagocytosis can stimulate the release of arachidonic acid (AA) metabolites, the possibility that secretion of these powerful mediators of inflammation might be induced by chrysotile asbestos was investigated in vitro. Rat AM were treated in vitro with chrysotile asbestos, and the cyclooxygenase products--prostaglandins, thromboxane B2 (TXB2), 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT)--and lipoxygenase products--leukotrienes (LT), hydroxyeicosatetraenoic acids (HETE)--secreted in the medium were isolated by high-performance liquid chromatography. Composition of the AA metabolites released was compared with that from those stimulated by the calcium ionophore A 23187 (20 microM) and by another particulate phagocytic stimulus, i.e., carbonyl iron beads. Calcium ionophore stimulation induced a marked release of various AA metabolites in the medium from both the cyclooxygenase pathway (HHT, TXB2, and PGE2, in decreasing quantities, respectively) and the lipoxygenase pathway (LTB4, 5-HETE, 12-HETE, and LTC4). The major product was LTB4. Treatment of the macrophages with asbestos fibers induced the release of a similar array of AA metabolites, although there were smaller amounts of LTC4 and 12-HETE, but increased quantities of PGF2 alpha. A time course study showed a steady increase in metabolite production for 1 h, followed by a plateau. In addition, the amount of metabolites released was dependent on asbestos concentrations. Phagocytosis of iron beads induced the secretion of the same metabolites as asbestos stimulation, but in larger quantities, probably reflecting the lack of cytotoxicity of the particle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different sensitivities of rat and human red cells to exogenous Ca2+

During an examination of the effects of shear and of the Ca2+ ionophore A23187 on Ca2+ entry into erythrocytes of rats and humans, we noted that rat erythrocytes were much more sensitive to Ca2+-induced hemolysis than the human cells. An examination of the effect of Ca2+ on transglutaminase, a cytosolic enzyme in the erythrocyte which cross-links membrane proteins and renders cells less deformable, demonstrated a correlation between enzyme activity and Ca2+-induced hemolysis. Both rat and human cells subjected to shear-induced Ca2+ entry exhibited increased enzyme activity and altered membrane protein SDS-PAGE patterns. Twenty micromolar A23187 with Ca2+ at concentrations above 80 microM caused hemolysis of rat erythrocytes. In contrast to human erythrocytes, under these conditions no membranes were recoverable from rat erythrocytes. At lower concentrations of Ca2+ (25 and 50 microM), however, rat erythrocytes maintained integrity, and exhibited enhanced transglutaminase activity and cross-linking of membrane proteins. The rat enzyme can be activated 30% by 10 microM Ca2+, while 50 microM Ca2+ was necessary to achieve a similar activation of the enzyme from human red blood cells. In studies of shear-stimulated Ca2+ uptake by erythrocytes the rat red cell enzyme was more readily activated. The SDS-PAGE pattern of rat red cell membranes after a 30 sec shear showed specific changes in protein banding, including the appearance of bands greater than 330 kDa. Changes in protein banding were also apparent in cytosolic proteins. This work supports the view that shear-induced Ca2+ entry activates transglutaminase that leads to cross-linking of membrane components, a loss of cell integrity, and eventual cell death.     

β-连环蛋白在高糖诱导的内皮细胞损伤中的作用

内皮细胞功能紊乱被认为是高糖诱导血管并发症的始动因素和加重的基础。Wnt／β-连环蛋白信号途径在内皮细胞增生和凋亡的调控上起着重要作用,这条信号通路关键调节靶点是胞浆中β-连环蛋白的水平,它决定了Wnt靶基因的活化水平。当内皮细胞处于高糖环境中,β-连环蛋白减少,Wnt信号减弱,内皮细胞抗凋亡能力下降,增殖受到抑制,从而导致血管并发症。除此以外,在高糖作用下,β-连环蛋白介导的内皮细胞间黏附连接的破坏将导致内皮通透性增高,使蛋白质等大分子物质漏出血管外,同样造成血管功能紊乱。     

Mechanism of Ca(2+) overload in endothelial cells exposed to simulated ischemia

OBJECTIVE: Several studies have shown that myocardial ischemia leads to functional failure of endothelial cells (EC) whereby disturbance of Ca(2+) homeostasis may play an important role. The mechanisms leading to Ca(2+) disbalance in ischemic EC are not fully understood. The aim of this study was to test effects of different components of simulated ischemia (glucose deprivation, anoxia, low extracellular pH (pH(o)) and lactate) on Ca(2+) homeostasis in EC. METHODS: Cytosolic Ca(2+) (Ca(i)), cytosolic pH (pH(i)) and ATP content were measured in cultured rat coronary EC. RESULTS: In normoxic cells 60 min glucose deprivation at pH(o) 7.4 had no effect on pH(i). It only slightly increased Ca(i) and decreased ATP content. Reduction of pH(o) to 6.5 under these conditions led to marked cytosolic acidosis and Ca(i) overload, but had no effect on ATP content. Anoxia at pH(o) 6.5 had no additional effect on Ca(i) overload, but significantly reduced cellular ATP. Addition of 20 mmol/l lactate to anoxia at pH(o) 6.5 accelerated Ca(i) overload due to faster cytosolic acidification. Acidosis-induced Ca(i) overload was prevented by inhibition of Ca(2+) release channels of endoplasmic reticulum (ER) with 3 micromol/l ryanodine or by pre-emptying the ER with thapsigargin. Re-normalisation of pH(o) for 30 min led to recovery of pH(i), but not of Ca(i). CONCLUSION: The ischemic factors leading to cytosolic acidosis (low pH(o) and lactate) cause Ca(i) overload in endothelial cells, while anoxia and glucose deprivation play only a minor role. The ER is the main source for this Ca(i) rise. Ca(i) overload is not readily reversible.     

Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit

We show here that mouse pluripotent hematopoietic stem cells can be maintained in vitro on stroma for at least 3 weeks at levels close to those found in bone marrow. The extent of stem cell maintenance is affected by the nature of the stromal cells. The stromal cell line S17 supported stem cells significantly better than heterogeneous, primary stromal layers or the stromal cell line Strofl-1. Stem cells cultured on S17 repopulated all hematopoietic lineages in marrow-ablated hosts for at least 10 months, indicating that this culture system maintained primitive stem cells with extensive proliferative capacity. Furthermore, we demonstrate that, while pluripotent stem cells express c-kit, this receptor appears to play only a minor role in stem cell maintenance in vitro. The addition of an antibody that blocks the interaction of c-kit with its ligand essentially abrogated myelopoiesis in cultures. However, the level of stem cells in antibody-treated cultures was similar to that found in untreated cultures. Thus, it seems likely that the maintenance of primitive stem cells in vitro depends on yet unidentified stromal cell-derived factor(s).     

Suppression of DNA synthesis in hepatoma cells exposed to glucocorticoid hormone in vitro

Glucocorticoid hormone is shown to markedly suppress DNA synthesis in a line of rat hepatoma cells in vitro. In the presence of 300 nM hydrocortisone or 30 nM dexamethasone the incorporation of radioactive thymidine falls to 50% of control levels by 36 hr, and at higher concentrations of hormone inhibition can be noted as early as 12 hr and is nearly complete by 24 hr. This inhibition of radioactive thymidine incorporation reflects a true suppression of DNA synthesis, is accompanied by a corresponding inhibition of cell proliferation, and is readily reversible upon subsequent removal of hormone. In contrast to previously described effects of the glucocorticoid hormones on various cells of lymphoid origin, the inhibition of DNA synthesis in these hepatoma cells is not accompanied by appreciable cell lysis or by degradation of preformed DNA, and even when [(3)H]thymidine incorporation into DNA is inhibited by 90% or more, incorporation of [(14)C]uridine into RNA proceeds with little change. These findings all parallel previous observations on the effects of glucocorticoid hormone on the livers of intact animals and suggest that studies on the mechanism of the inhibition of DNA synthesis in the present more isolated system may lead to a better understanding of the means by which these compounds inhibit liver growth in vivo.Despite the ready suppressibility of DNA synthesis in these hepatoma cells and in two other cell lines of liver origin, none of these cell lines was found to be inducible for tyrosine aminotransferase. The apparent dissociation between two "steroid-sensitive" phenomena is of interest and warrants further investigation.     

DNA damage and mutation in human cells exposed to nitric oxide in vitro.

Nitric oxide (NO.) is a physiological messenger formed by several cell types. Reaction with O2 forms oxides that nitrosate amines at pH values near 7. We now report experiments in which NO. was added to intact human cells and to aerobic solutions of DNA, RNA, guanine, or adenine. TK6 human lymphoblastoid cells were mutated 15- to 18-fold above background levels at both the HPRT and TK gene loci. Xanthine and hypoxanthine, from deamination of guanine and adenine, respectively, were formed in all cases. NO. induced dose-responsive DNA strand breakage. Yields of xanthine ranged from nearly equal to about 80-fold higher than those of hypoxanthine. Yields of xanthine and hypoxanthine from nucleic acids were higher than those from free guanine and adenine. This was most pronounced for xanthine; 0.3 nmol/mg was formed from free guanine vs. 550 nmol/mg from calf thymus RNA. Nitric oxide added to TK6 cells produced a 40- to 50-fold increase in hypoxanthine and xanthine in cellular DNA. We believe that these results, plus the expected deaminations of cytosine to uracil and 5-methylcytosine to thymine, account for the mutagenicity of nitric oxide toward bacteria and mammalian cells.     

Increased susceptibility of microcytic red blood cells to in vitro oxidative stress

Abstract: Oxidative damage to erythrocytes in thalassaemia has been related to generation of free radicals by an excess of denaturated α- or β-globin chains, intracellular iron overload and low concentration of normal haemoglobin (HGB). Two good indicators of such oxidative damage are the high red blood cell (RBC) malonyldialdehyde (MDA) production detected following exogenous oxidant stress and the decrease of pyrimidine 5′-nucleotidase (P5N), the most sensitive enzyme to SH-group damage in vivo. Conflicting data, however, have so far accumulated in the literature concerning differences in oxidative damage between the different forms of thalassaemia and iron deficiency anaemia (IDA). In the present study, oxidative susceptibility, as defined by the production of MDA in vitro and antioxidant capacity, as measured by the activity of RBC glutathione peroxidase (GPx), superoxide dismutase (SOD) and by reduced glutathione (GSH), have been studied in microcytic RBCs from patients with β-thalassaemia trait, Spanish (δβ)°-thalassaemia heterozygotes (δβ-thalassaemia trait) and iron deficiency anaemia (IDA). The results are consistent with the existence of significant differences in the severity and pattern of oxidative stress susceptibility between β-thalassaemia trait (increased MDA production and higher SOD and GPx activities) and the other two forms of microcytosis (δβ thalassaemia trait and IDA). Furthermore, the finding of normal P5′N activity in δβ thalassaemia trait, gives further support to the less intense peroxidative environment of RBCs in this form of thalassaemia when compared to β-thalassaemia trait, characterized by acquired RBC P5′N deficiency due to oxidative damage.     

Role of protein kinase C-angiotensin II pathway for extracellular matrix production in cultured human mesangial cells exposed to high glucose levels

The intrarenal renin-angiotensin system has been implicated in the pathogenesis of diabetic nephropathy. This study investigates the mechanisms for glucose-induced increase in angiotensin II (AII) production by human mesangial cells (MCs) in relation to protein kinase C (PKC). We also examine whether locally produced AII mediates extracellular matrix protein production in high-glucose conditions. Human MCs were cultured in 5 or 33 mmol/l glucose for 8 days, and were incubated with or without 5 mmol/l GFX, a PKC inhibitor, 0.1 micromol/l candesartan cilexetil (CC), a specific type 1 AII receptor antagonist, for another 24 h. In addition, MCs grown in 5 mmol/l glucose were incubated with 0.1 micromol/l phorbol-12,13-dibutyrate (PDBu) for 24 h. AII, TGF-beta1, fibronectin and type IV collagen in the culture media were measured by ELISA. The amount of AII secreted from MCs exposed to high-glucose levels was significantly greater (P<0.01) than that in normal glucose levels. The increase in AII production was completely prevented by GFX. The addition of PDBu mimicked the effect of glucose on AII production. The glucose-induced increases in the production of TGF-beta1, fibronectin and type IV collagen were partially, but significantly restored (P<0.01) by CC, while GFX totally abolished these effects of glucose. These results suggest that elevated glucose levels stimulate AII production via mechanisms dependent on glucose-induced PKC activation in human MCs, and that locally produced AII partly mediates the increase in mesangial matrix synthesis in high-glucose conditions.     

K:CI cotransport in red cells of transgenic mice expressing high levels of human hemoglobin S

K:CI cotransport is involved in generating dense red blood cells (RBCs) in homozygotes for HbS (SS). We report on the properties of this transport system in RBCs from control and transgenic mice expressing high levels of human α^H and β^S chains. Unlike human SS RBCs, mouse RBCs incubated in isotonic media exhibited a CI⁻-dependent K⁺ efflux and therefore have a different set-point for activation. This basal efflux was slightly stimulated by cell swelling to values five times smaller than that in human SS cells; In addition, the delay time for activation was shorter in transgenic than in control mice, but fourtold longer than that of human SS cells. These properties cast doubt on the physiological impact of the mouse K:CI cotransporter on RBC volume regulation in the mouse and suggest that there are intrinsic differences between the human K:CI cotransporter and the putative transporter in mice. Am. J. Hematol. 55:112-114, 1997. © 1997 Wiley-Liss, Inc.     

High prevalence of prediabetes and diabetes in a population exposed to high levels of an organochlorine cocktail

Aims/hypothesis

A heavily polluted area of Eastern Slovakia was targeted by the PCBRISK cross-sectional survey to search for possible links between environmental pollution and both prediabetes and diabetes.

Methods

Associations of serum levels of five persistent organic pollutants (POPs), namely polychlorinated biphenyls (PCBs), 2,2′-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p′-DDE), 2,2′-bis(4-chlorophenyl)-1,1,1-trichloro-ethane (p,p′-DDT), hexachlorobenzene (HCB) and β-hexachlorocyclohexane (β-HCH), with prediabetes and diabetes were investigated in 2,047 adults. Diabetes and prediabetes were diagnosed by fasting plasma glucose in all participants and by OGTT in 1,220 compliant participants.

Results

Our population was stratified in terms of individual POPs quintiles and associations between environmental pollution, prediabetes and diabetes were investigated. Prevalence of prediabetes and diabetes increased in a dose-dependent manner, with individuals in upper quintiles of individual POPs showing striking increases in prevalence of prediabetes as shown by OR and 95% CI for PCBs (2.74; 1.92–3.90), DDE (1.86; 1.17–2.95), DDT (2.48; 1.77–3.48), HCB (1.86; 1.7–2.95) and β-HCH (1.97; 1.28–3.04). Interestingly, unlike PCBs, DDT and DDE, increased levels of HCB and β-HCH seemed not to be associated with increased prevalence of diabetes. Nevertheless, individuals in the 5th quintile of the variable expressing the cumulative effect of all five POPs (sum of orders) had a more than tripled prevalence of prediabetes and more than six times higher prevalence of diabetes when compared with the 1st referent quintile.

Conclusions/interpretation

Increasing serum concentrations of individual POPs considerably increased prevalence of prediabetes and diabetes in a dose-dependent manner. Interaction of industrial and agricultural pollutants in increasing prevalence of prediabetes or diabetes is likely.