Natural antioxidants improve red blood cell "survival" in non-leukoreduced blood samples.

BACKGROUND: Blood collected in an anticoagulant can be kept refrigerated in an unmodified state within 5 - 6 weeks. Oxidative damage is considered to be a one of the major factors contributing to the development of storage lesions. Lipid and membrane proteins oxidation results in changes in cation gradients that affect the cell survival. AIM: In the present study we used the natural antioxidants and ion channels blockers (L-carnosine, spermine, phloretin and their mixtures) to prolong "survival" of red blood cells (RBCs), measured as the lack of PS exposure and cell hemolysis, in the Alsever's preservative solution upon hypothermic storage. RESULTS: We show that the mixture of carnosine (20 mM), spermine (20 µM) and phloretin (100 µM) effectively blunted phosphatidylserine (PS) exposure, Ca(2+) accumulation and RBCs hemolysis in non-leukoreduced low (∼ 2%) hematocrit samples after 36 days of storage as well as after 1 day of post-storage incubation of the stored cells in physiological saline solution. In addition, a slight but significant decrease in PS exposure was observed in non-leukoreduced high (∼ 20%) hematocrit samples after 36 days of storage with the mixture of substances. CONCLUSION: We conclude that the use of the mixture of natural antioxidants (carnosine, spermine, and phloretin) as an additive to blood preservative solution provides better RBCs storage and "survival".

Niflumic acid affects store-operated Ca(2+)-permeable (SOC) and Ca (2+)-dependent K (+) and Cl (-) ion channels and induces apoptosis in K562 cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells. However, the precise mechanisms by which NSAIDs facilitate apoptosis in tumor cells are not clear. In the present study, we show that niflumic acid (NA), a member of the fenamates group of NSAIDs and Cl(-) and Ca(2+)-activated Cl(-) (CAC) channels blocker, induced apoptosis (by ~8 %, 24 h treatment) and potentiated (by 8-10 %) apoptotic effect of endoplasmic reticulum Ca(2+) mobilizer thapsigargin (Tg) in human erythroleukemic K562 cell line. The whole-cell patch clamp and Fluo-3 flow cytometric experiments confirmed an inhibitory effect of NA (100 and 300 µM) on store-operated (SOC) channels. We also found that NA-blocked CAC channels were activated by acute application of Tg (2 µM) in K562 cells. NA blockage of CAC channels was accompanied by activation of Ca(2+)-activated K(+) (SK4) channels. The observed effects of NA were not connected with COX-2 inhibition since 100-nM NA (IC50 for COX-2 inhibition) did not induce either apoptosis or affect the channels activity. We conclude that inhibition of SOC channels plays a major role in NA-induced apoptosis. Increased apoptotic levels in Tg-treated K562 cells in the presence of NA may be due to the blockage of CAC and stimulation of SK4 channels in addition to SOC channels inhibition.

Dimethyl sulfoxide at high concentrations inhibits non-selective cation channels in human erythrocytes.

Dimethyl sulfoxide (DMSO), a by-product of the pulping industry, is widely used in biological research, cryobiology and medicine. On cellular level DMSO was shown to suppress NMDA-AMPA channels activation, blocks Na+ channel activation and attenuates Ca2+ influx (Lu and Mattson 2001). In the present study we explored the whole-cell patch-clamp to examine the acute effect of high concentrations of DMSO (0.1-2 mol/l) on cation channels activity in human erythrocytes. Acute application of DMSO (0.1-2 mol/l) dissolved in Cl--containing saline buffer solution significantly inhibited cation conductance in human erythrocytes. Inhibition was concentration-dependent and had an exponential decay profile. DMSO (2 mol/l) induced cation inhibition in Cl-- containing saline solutions of: 40.3 ± 3.9% for K+, 35.4 ± 3.1% for Ca2+ and 47.4 ± 1.9% for NMDG+. Substitution of Cl- with gluconate- increased the inhibitory effect of DMSO on the Na+ current. Inhibitory effect of DMSO was neither due to high permeability of erythrocytes to DMSO nor to an increased tonicity of the bath media since no effect was observed in 2 mol/l glycerol solution. In conclusion, we have shown that high concentrations of DMSO inhibit the non-selective cation channels in human erythrocytes and thus protect the cells against Na+ and Ca2+ overload. Possible mechanisms of DMSO effect on cation conductance are discussed.

Effect of chloride channel inhibitors on cytosolic Ca2+ levels and Ca2+-activated K+ (Gardos) channel activity in human red blood cells.

DIDS, NPPB, tannic acid (TA) and AO1 are widely used inhibitors of Cl(-) channels. Some Cl(-) channel inhibitors (NPPB, DIDS, niflumic acid) were shown to affect phosphatidylserine (PS) scrambling and, thus, the life span of human red blood cells (hRBCs). Since a number of publications suggest Ca(2+) dependence of PS scrambling, we explored whether inhibitors of Cl(-) channels (DIDS, NPPB) or of Ca(2+)-activated Cl(-) channels (DIDS, NPPB, TA, AO1) modified intracellular free Ca(2+) concentration ([Ca(2+)]i) and activity of Ca(2+)-activated K(+) (Gardos) channel in hRBCs. According to Fluo-3 fluorescence in flow cytometry, a short treatment (15 min, +37 °C) with Cl(-) channels inhibitors decreased [Ca(2+)]i in the following order: TA > AO1 > DIDS > NPPB. According to forward scatter, the decrease of [Ca(2+)]i was accompanied by a slight but significant increase in cell volume following DIDS, NPPB and AO1 treatments. TA treatment resulted in cell shrinkage. According to whole-cell patch-clamp experiments, TA activated and NPPB and AO1 inhibited Gardos channels. The Cl(-) channel blockers further modified the alterations of [Ca(2+)]i following ATP depletion (glucose deprivation, iodoacetic acid, 6-inosine), oxidative stress (1 mM t-BHP) and treatment with Ca(2+) ionophore ionomycin (1 µM). The ability of the Cl(-) channel inhibitors to modulate PS scrambling did not correlate with their influence on [Ca(2+)]i as TA and AO1 had a particularly strong decreasing effect on [Ca(2+)]i but at the same time enhanced PS exposure. In conclusion, Cl(-) channel inhibitors affect Gardos channels, influence Ca(2+) homeostasis and induce PS exposure of hRBCs by Ca(2+)-independent mechanisms.

Inhibitory effect of furosemide on non-selective voltage-independent cation channels in human erythrocytes.

BACKGROUND: Furosemide, a loop diuretic inhibiting the renal tubular Na(+),K(+),2Cl(-) cotransporter, has been shown to decrease cytosolic Ca(2+) concentration ([Ca(2+)](i)) in platelets and erythrocytes. [Ca(2+)](i) in erythrocytes is a function of Ca(2+) permeable cation channels. Activation of those channels e.g. by energy depletion or oxidative stress leads to increase of [Ca(2+)](i), which in turn triggers eryptosis, a suicidal erythrocyte death characterized by cell membrane scrambling. The present study was performed to explore whether furosemide influences the cation channels and thus influences eryptosis. METHODS: Cation channel activity was determined by whole-cell patch clamp, [Ca(2+)](i) utilizing Fluo3 fluorescence and annexin V binding to estimate cell membrane scrambling with phosphatidylserine exposure. RESULTS: A 45 min exposure to furosemide (10 and 100 µM) slightly, but significantly decreased cation channel activity and [Ca(2+)](i) in human erythrocytes drawn from healthy individuals. ATP-depletion (> 3 hours, +37°C, 6 mM ionosine and 6 mM iodoacetic acid) enhanced the non-selective cation channel activity, increased [Ca(2+)](i) and triggered cell membrane scrambling, effects significantly blunted by furosemide (10 - 100 µM). Oxidative stress by exposure to tert-butylhydroperoxide (0.1 -1 mM) similarly enhanced the non-selective cation channels activity, increased [Ca(2+)](i) and triggered cell membrane scrambling, effects again significantly blunted by furosemide (10 - 100 µM). CONCLUSIONS: The present study shows for the first time that the loop diuretic furosemide applied at micromolar concentrations (10 - 100 µM) inhibits non-selective cation channel activity in and eryptosis of human erythrocytes.

Decreased redox-sensitive erythrocyte cation channel activity in aquaporin 9-deficient mice.

Survival of the malaria pathogen Plasmodium falciparum in host erythrocytes requires the opening of new permeability pathways (NPPs) in the host cell membrane, accomplishing entry of nutrients, exit of metabolic waste products such as lactate and movement of inorganic ions such as Cl⁻, Na⁺ and Ca²âº. The molecular identity of NPPs has remained largely elusive but presumably involves several channels, which partially can be activated by oxidative stress in uninfected erythrocytes. One NPP candidate is aquaporin 9 (AQP9), a glycerol-permeable water channel expressed in erythrocytes. Gene-targeted mice lacking functional AQP9 (aqp⁻/⁻) survive infection with the malaria pathogen Plasmodium berghei better than their wild-type littermates (aqp9⁺/⁺). In the present study whole-cell patch-clamp recordings were performed to explore whether ion channel activity is different in erythrocytes from aqp⁻/⁻ and aqp9⁺/⁺ mice. As a result, the cation conductance (K⁺ > Na⁺ > Ca²âº ≫ NMDG⁺) was significantly lower in erythrocytes from aqp⁻/⁻ than in erythrocytes from aqp9⁺/⁺ mice. Oxidative stress by exposure for 15-30 min to 1 mM H2O2 or 1 mM tert-butyl-hydroperoxide enhanced the cation conductance and increased cytosolic Ca²âº concentration, effects significantly less pronounced in erythrocytes from aqp⁻/⁻ than in erythrocytes from aqp9⁺/⁺ mice. In conclusion, lack of AQP9 decreases the cation conductance of erythrocytes, an effect that possibly participates in the altered susceptibility of AQP9-deficient mice to infection with P. berghei.

Effect of casein kinase 1α activator pyrvinium pamoate on erythrocyte ion channels.

Pharmacological modification of protein kinase CK1 (casein kinase 1) has previously been shown to influence suicidal erythrocyte death or eryptosis, which is triggered by activation of Cl(-)-sensitive Ca(2+)-permeable cation channels. Ca(2+) entering through those channels stimulates cell membrane scrambling and opens Ca(2+)-activated K(+)-channels resulting in KCl exit and thus cell shrinkage. The specific CK1-inhibitor D4476 (1 µM) blunted, whereas the specific CK1 αactivator pyrvinium pamoate (10 µM) enhanced cell membrane scrambling. The substances were at least partially effective through modification of cytosolic Ca(2+)-activity. The present study explored, whether pyrvinium pamoate indeed influences Cl(-)-sensitive cation-channels in erythrocytes. As a result, removal of Cl(-)increased Fluo3-fluorescence (reflecting cytosolic Ca(2+)-activity), triggered cell membrane scrambling (apparent from annexin-V-binding), and decreased forward scatter (pointing to cell shrinkage). Pyrvinium pamoate significantly augmented the effect of Cl(-)-removal on Fluo3 fluorescence and annexin-V-binding, but blunted the effect on forward scatter. According to whole cell patch clamp recording, Cl(-)removal activated a cation current, which was significantly enhanced by pyrvinium pamoate. Pyrvinium pamoate inhibited Ca(2+)-activated K(+)-channels. Ca(2+)-ionophore ionomycin (1 µM) decreased forward scatter, an effect significantly blunted by pyrvinium pamoate. In conclusion, pyrvinium pamoate activates Cl(-)-sensitive Ca(2+)-permeable cation channels with subsequent Ca(2+)-entry and inhibits Ca(2+)-activated K(+)-channels thus blunting the stimulating effect of Ca(2+) on those channels, K(+)-exit and thus cell shrinkage.

Dicoumarol activates Ca2+-permeable cation channels triggering erythrocyte cell membrane scrambling.

Dicoumarol, a widely used anticoagulant, may cause anemia, which may result from enhanced erythrocyte loss due to bleeding or due to accelerated erythrocyte death. Erythrocytes may undergo suicidal death or eryptosis, characterized by cell shrinkage and phospholipid scrambling of the cell membrane. Eryptosis may be triggered by increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)). The present study explored, whether dicoumarol induces eryptosis. [Ca(2+)](i) was estimated from Fluo3-fluorescence, cation channel activity utilizing whole cell patch clamp, cell volume from forward scatter, phospholipid scrambling from annexin-V-binding, and hemolysis from haemoglobin release. Exposure of erythrocytes for 48 hours to dicoumarol (=10 µM) significantly increased [Ca(2+)](i), enhanced cation channel activity, decreased forward scatter, triggered annexin-V-binding and elicited hemolysis. Following exposure to 30 µM dicoumarol, annexin-V-binding affected approximately 15%, and hemolysis 2% of treated erythrocytes. The stimulation of annexin-V-binding by dicoumarol was abrogated in the nominal absence of Ca(2+). In conclusion, dicoumarol stimulates suicidal death of erythrocytes by stimulating Ca(2+) entry and subsequent triggering of Ca(2+) dependent cell membrane scrambling.

Sphingosine but not sphingosine-1-phosphate stimulates suicidal erythrocyte death.

Sphingosine kinase 1 phosphorylates sphingosine, which is converted to ceramide by ceramide synthetase. Ceramide triggers eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. Erythrocytes lack sphingosine phosphate-degrading enzymes and thus store large quantities of sphingosine phosphate. The present study explored the influence of sphingosine and sphingosine phosphate on eryptosis. [Ca(2+)](i), was estimated from Fluo3 fluorescence, cell volume from forward scatter and PS exposure from annexin V-binding in FACS analysis. Sphingosine (0.1 - 10 µM) but not sphingosine-1- phosphate (0.1 - 10 µM) increased [Ca(2+)](i), decreased cell volume and increased PS-exposure. The observations disclose sphingosine, but not sphingosine-1-phosphate, as a strong inducer of eryptosis.

Beauvericin induced erythrocyte cell membrane scrambling.

Beauvericin is a mycotoxin with antiviral, antibacterial, nematicidal, insecticidal, cytotoxic, and apoptotic activity. Similar to nucleated cells erythrocytes may undergo suicidal death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. Eryptosis may be triggered by energy depletion leading to increase of cytosolic Ca²+ activity. The present study thus explored whether beauvericin is able to trigger eryptosis and influence eryptosis following energy depletion. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, cell volume from forward scatter in FACS analysis, cytosolic Ca²+ concentration from Fluo3 fluorescence, cytosolic ATP concentration from a luciferase-assay and ion channel activity with whole cell patch clamp. Exposure to beauvericin (≥ 5 µM) significantly decreased erythrocyte ATP concentration and increased cytosolic Ca²+ concentration as well as annexin V-binding. The effect of beauvericin on annexin V binding was significantly blunted by removal of extracellular Ca²+. Glucose depletion (48 h) was followed by, increase of Fluo3 fluorescence, decrease of forward scatter and increase of annexin V-binding. Beauvericin (≥ 1 µM) augmented the effect of glucose withdrawal on Fluo3 fluorescence and annexin V-binding, but significantly blunted the effect of glucose withdrawal on forward scatter, an effect paralleled by inhibition of Ca²+ activated K+ channels. The present observations disclose novel effects of beauvericin, i.e. stimulation of Ca²+ entry with subsequent cell membrane scrambling and inhibition of Ca²+ activated K+ channels with blunting of cell shrinkage.

Inhibition of cation channels in human erythrocytes by spermine.

In erythrocytes, spermine concentration decreases gradually with age, which is paralleled by increases of cytosolic Ca²+ concentration, with subsequent cell shrinkage and cell membrane scrambling. Cytosolic Ca²+ was estimated from fluo-3 fluorescence, cell volume from forward scatter, cell membrane scrambling from annexin V binding and cation channel activity with whole-cell patch-clamp in human erythrocytes. Extracellular spermine exerted a dual effect on erythrocyte survival. At 200 µM spermine blunted the increase of intracellular Ca²+, cell shrinkage and annexin V binding following 48 h exposure of cells at +37 °C. In contrast, short exposure (10-30 min) of cells to 2 mM spermine was accompanied by increased cytosolic Ca²+ and annexin binding. Intracellular addition of spermine at subphysiological concentration (0.2 µM) significantly decreased the conductance of monovalent cations (Na+, K+, NMDG+) and of Ca²+. Moreover, spermine (0.2 µM) blunted the stimulation of voltage-independent cation channels by Cl⁻ removal. Spermine (0.2 and 200 µM) added to the extracellular bath solution similarly inhibited the cation conductance in Cl⁻-containing bath solution. The effect of 0.2 µM spermine, but not the effect of 200 µM, was rapidly reversible. Acute addition (250 µM) of a naphthyl acetyl derivative of spermine (200 µM) again significantly decreased basal cation conductance in NaCl bath solution and inhibited voltage-independent cation channels. Spermine is a powerful regulator of erythrocyte cation channel cytosolic Ca²+ activity and, thus, cell survival.

Blunted IgE-mediated activation of mast cells in mice lacking the serum- and glucocorticoid-inducible kinase SGK3.

Previous studies have shown that pharmacological inhibition of the phosphoinositol-3 (PI3) kinase disrupts the activation of mast cells. Through phosphoinositide-dependent kinase PDK1, PI3 kinase activates the serum- and glucocorticoid-inducible kinase 3 (SGK3). The present study explored the role of SGK3 in mast cell function. Mast cells were isolated and cultured from bone marrow (BMMCs) of gene-targeted mice lacking SGK3 (sgk3(-/-)) and their wild-type littermates (sgk3(+/+)). BMMC numbers in the ear conch were similar in both genotypes. Stimulation with IgE and cognate antigen triggered the release of intracellular Ca(2+) and entry of extracellular Ca(2+). Influx of extracellular Ca(2+) but not Ca(2+) release from intracellular stores was significantly blunted in sgk3(-/-) BMMCs compared with sgk3(+/+) BMMCs. Antigen stimulation further led to a rapid increase of a K(+)-selective conductance in sgk3(+/+) BMMCs, an effect again blunted in sgk3(-/-) BMMCs. In contrast, the Ca(2+) ionophore ionomycin activated K(+) currents to a similar extent in sgk3(-/-) and in sgk3(+/+) BMMCs. ß-Hexosaminidase release, triggered by antigen stimulation, was also significantly decreased in sgk3(-/-) BMMCs. IgE-dependent anaphylaxis measured as a sharp decrease in body temperature upon injection of DNP-HSA antigen was again significantly blunted in sgk3(-/-) compared with sgk3(+/+) mice. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk3(-/-) than in sgk3(+/+) mice. In conclusion, both in vitro and in vivo function of BMMCs are impaired in gene targeted mice lacking SGK3. Thus SGK3 is critical for proper mast cell function.

Increased cation conductance in human erythrocytes artificially aged by glycation.

Excessive glucose concentrations foster glycation and thus premature aging of erythrocytes. The present study explored whether glycation-induced erythrocyte aging is paralleled by features of suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface and cell shrinkage. Both are triggered by increases of cytosolic Ca(2+) concentration ([Ca(2+)](i)), which may result from activation of Ca(2+) permeable cation channels. Glycation was accomplished by exposure to high glucose concentrations (40 and 100 mM), phosphatidylserine exposure estimated from annexin binding, cell shrinkage from decrease of forward scatter, and [Ca(2+)](i) from Fluo3-fluorescence in analysis via fluorescence-activated cell sorter. Cation channel activity was determined by means of whole-cell patch clamp. Glycation of total membrane proteins, immunoprecipitated TRPC3/6/7, and immunoprecipitated L-type Ca(2+) channel proteins was estimated by Western blot testing with polyclonal antibodies used against advanced glycation end products. A 30-48-h exposure of the cells to 40 or 100 mM glucose in Ringer solution (at 37 degrees C) significantly increased glycation of membrane proteins, hemoglobin (HbA(1c)), TRPC3/6/7, and L-type Ca(2+) channel proteins, enhanced amiloride-sensitive, voltage-independent cation conductance, [Ca(2+)](i), and phosphatidylserine exposure, and led to significant cell shrinkage. Ca(2+) removal and addition of Ca(2+) chelator EGTA prevented the glycation-induced phosphatidylserine exposure and cell shrinkage after glycation. Glycation-induced erythrocyte aging leads to eryptosis, an effect requiring Ca(2+) entry from extracellular space.

Enhanced eryptosis of erythrocytes from gene-targeted mice lacking annexin A7.

Annexin A7 is a ubiquitously expressed Ca(2+)- and phospholipid-binding protein. Erythrocytes from mice lacking annexin A7 (anxA7(-/-)) are deformed and relatively resistant to osmotic swelling. In normal erythrocytes, hyperosmotic shock, Cl(-) removal, and energy depletion (glucose removal) trigger PGE(2) formation, which stimulates Ca(2+)-permeable cation channels, increases cytosolic Ca(2+) activity ([Ca(2+)](i)), and thus triggers suicidal death of erythrocytes or eryptosis, characterized by scrambling of the cell membrane with phosphatidylserine exposure at the cell surface. The present experiments explored the influence of annexin A7 deficiency on eryptosis. In erythrocytes from annexin A7-deficient mice (anxA7(-/-)) and wild-type mice (anxA7(+/+)), PGE(2) formation was determined utilizing an immunoassay, ion channel activity by whole-cell patch clamp recording, [Ca(2+)](i) by fluo3 fluorescence, and phosphatidylserine exposure by binding of annexin A5 in fluorescence activated cell sorter (FACS) analysis. Erythrocyte number and hematocrit were significantly smaller in blood from anx7(-/-) than in anx7(+/+) mice. Cl(-)-removal (replacement with gluconate) stimulated PGE(2)-formation, activated cation currents, increased [Ca(2+)](i), and triggered phosphatidylserine exposure, effects significantly more pronounced in anx7(-/-) than in anx7(+/+) erythrocytes. Hyperosmotic shock (addition of 400 mM sucrose) and glucose depletion (removal of glucose) similarly increased cytosolic Ca(2+) activity and triggered phosphatidylserine exposure, effects again significantly more pronounced in anx7(-/-) than in anx7(+/+) erythrocytes. The effects of Cl(-) removal on PGE(2) formation and the cation current, as well as the effect of hypertonic cell shrinkage on [Ca(2+)](i) and cell membrane scrambling, were blunted following inhibition of cyclooxygenase by aspirin or diclofenac. In conclusion, lack of annexin A7 sensitizes the erythrocytes for "proapoptotic" Ca(2+) overload, an effect shortening the life span of the affected erythrocytes and, thus, leading to anemia.

Acid-sensitive outwardly rectifying anion channels in human erythrocytes.

Acid-sensitive outwardly rectifying anion channels (ASOR) have been described in several mammalian cell types. The present whole-cell patch-clamp study elucidated whether those channels are expressed in erythrocytes. To this end whole-cell recordings were made in human erythrocytes from healthy donors treated with low pH and high osmotic pressure. When the pipette solution had a reduced Cl(-) concentration, treatment of the cells with Cl(-)-containing normal and hyperosmotic (addition of sucrose and polyethelene glycol 1000 [PEG-1000] to the Ringer) media with low pH significantly increased the conductance of the cells at positive voltages. Channel activity was highest in the PEG-1000 media (95 and 300 mM PEG-1000, pH 4.5 and 4.3, respectively) where the current-voltage curves demonstrated strong outward rectification and reversed at -40 mV. Substitution of the Cl(-)-containing medium with Cl(-)-free medium resulted in a decrease of the conductance at hyperpolarizing voltages, a shift in reversal potential (to 0 mV) and loss of outward rectification. The chloride currents were inhibited by chloride channels blockers DIDS and NPPB (IC(50) for both was approximately 1 mM) but not with niflumic acid and amiloride. The observations reveal expression of ASOR in erythrocytes.

Modulation of suicidal erythrocyte cation channels by an AMPA antagonist.

In neurons alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are heteromeric cation channels composed of different sub-units, including GluA1-GluA4. When expressed without GluA2, AMPA receptors function as Ca(2+)-permeable cation channels. In erythrocytes, activation of Ca(2+)-permeable cation channels triggers suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with subsequent exposure of phosphatidylserine at the cell surface. Activators of the channels and thus eryptosis include removal of extracellular Cl(-) (replaced by gluconate) and energy depletion (removal of glucose). The present study explored whether GluA1 is expressed in human erythrocytes and whether pharmacological AMPA receptor inhibition modifies Ca(2+) entry and suicidal death of human erythrocytes. GluA1 protein abundance was determined by confocal microscopy, phosphatidylserine exposure was estimated from annexin V binding, cell volume from forward scatter in FACS analysis, cytosolic Ca(2+) concentration from Fluo3 fluorescence and channel activity by whole-cell patch-clamp recordings. As a result, GluA1 is indeed expressed in the erythrocyte cell membrane. The AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) inhibited the cation channels following Cl(-) removal and the eryptosis following Cl(-) removal or energy depletion. The present study reveals a novel action of AMPA receptor antagonists and raises the possibility that GluA1 or a pharmacologically related protein participates in the regulation of Ca(2+) entry into and suicidal death of human erythrocytes.

Inhibition of cation channels and suicidal death of human erythrocytes by zidovudine.

Zidovudine, a drug widely used in the treatment of AIDS, has been shown to influence cytosolic calcium activity in HIV-infected lymphocytes. Thus, zidovudine may modify the activity of Ca(2+)-permeable ion channels. In erythrocytes, activation of Ca(2+)-permeable cation channels stimulates eryptosis, the suicidal erythrocyte death. Eryptosis is characterized by cell shrinkage (apparent from a decrease of forward scatter) and phosphatidylserine (PS) exposure (apparent from annexin V-binding) at the erythrocyte surface. Triggers of eryptosis include isotonic cell shrinkage (Cl(-) replacement by gluconate), energy depletion (removal of glucose) or exposure to a variety of drugs including azathioprine. The present study explored, whether zidovudine influences the activity of erythrocytic Ca(2+)-permeable cation channels and eryptosis. Whole-cell patch-clamp recordings indeed revealed that zidovudine blocked the Ca(2+)-permeable cation channels activated by Cl(-) removal. In the presence of Cl(-) and glucose, the percentage of annexin V-binding cells was low and not significantly modified by the presence of zidovudine. Both, Cl(-) removal and glucose depletion increased annexin V-binding and decreased forward scatter, effects significantly blunted by zidovudine (2 microg/ml). According to Fluo3 fluorescence, zidovudine (2 microg/ml) did not significantly modify cytosolic Ca(2+) concentration under control conditions, but significantly blunted the increase in cytosolic Ca(2+) activity following glucose depletion. Furthermore, zidovudine significantly inhibited azathioprine-induced eryptosis. The present observations disclose a completely novel effect of zidovudine, i.e. its inhibitory influence on Ca(2+) entry and subsequent suicidal erythrocyte death during isotonic cell shrinkage or energy depletion.

Effect of peroxynitrite on passive K+ transport in human red blood cells.

Peroxynitrite is generated in vivo by the reaction between nitric oxide, from endothelial and other cells, and the superoxide anion. It is therefore pertinent to examine its effects on the membrane permeability of red blood cells. Treatment of human red blood cells with peroxynitrite (nominally 1 mM) markedly stimulated passive K+ permeability. The main effect was on a Cl(-)-independent K+ pathway, which remains unidentified. Although K+-Cl- cotransport (KCC) was stimulated, this was dependent on saline composition, being inhibited by physiological levels of glucose (IC50 4 mM), and also by sucrose and MOPS. Effects on the Cl(-)-independent K+ pathway were less dependent on saline composition, and were not inhibited by amiloride, ethylisopropylamiloride, dimethylamiloride or gadolinium. Na+-K+-2Cl- cotransporter was inhibited whilst there was little effect on the Gardos channel (Ca2+-activated K+ channel). Peroxynitrite was markedly more effective in oxygenated cells than deoxygenated ones. Treatment with peroxynitrite per se did not affect initial cell volume. Anisotonic swelling modestly increased the Cl(-)-independent K+ influx, but did not affect peroxynitrite-stimulated KCC. Decreasing extracellular pH from 7.4 to 7.2 or 7.0 increased KCC stimulation, whilst the Cl(-)-independent component of K+ transport was lowest at pH 7.2. Finally, protein phosphatase inhibition with calyculin A (100 nM) inhibited KCC, implying that, as with other KCC stimuli, peroxynitrite acts via decreased protein phosphorylation; pre-treatment with calyculin A also inhibited the Cl(-)-independent component of K+ transport. These findings are relevant to the actions of peroxynitrite in vivo.

Effect of the ionic strength and prostaglandin E2 on the free Ca2+ concentration and the Ca2+ influx in human red blood cells.

Human red blood cells (RBCs) were loaded with the Ca(2+)-sensitive fluorescent dye fura-2 to investigate the effects of media ionic strength and prostaglandin E2 (PGE2) on the intracellular free Ca2+ concentration ([Ca2+]i). [Ca2+]i of intact RBCs in a Ca(2+)-containing physiological (high) ionic strength (HIS) solution was 75.1 +/- 8.3 nM after 5 min incubation, increasing to 114.9 +/- 9.6 nM after 1 h. In Ca(2+)-containing low ionic strength (LIS) solutions, [Ca2+]i was significantly lower than in the Ca(2+)-containing HIS solution (p = 0.041 or 0.0385 for LIS solutions containing 200 or 250 mM sucrose, respectively), but, as in HIS solution, an increase of [Ca2+]i was seen after 1 h. In Ca(2+)-free (0 Ca2+ plus 15 microM EGTA) media, [Ca2+]i decreased (ranging from 15 to 21 nM), but were not significantly different in HIS or LIS, and did not change following 1 h incubation. The effect of the ionic strength and PGE2 on passive Ca2+ influx was investigated on ATP-depleted RBCs. Ca2+ influx was faster during the initial 10 min in comparison with the subsequent time period (10-45 min), both in HIS and LIS media, decreasing from 20.3 +/- 1.9 to 12.9 +/- 1.3 micromol/(lcells x h) in HIS, and from 36.7 +/- 5.3 to 8.6 +/- 1.2 micromol/(lcells x h) in LIS. Prostaglandin E2 (PGE2; 10(-7)-10(-11) M), dissolved in deionised water or in ethanol, did not affect [Ca2+]i in either normal or in ATP-depleted RBCs suspended in Ca(2+)-containing HIS medium. Finally, the addition of carbachol (100 microM) did not affect [Ca2+]i. The present findings suggest that stimulation of the Ca(2+)-activated K+ channel by PGE2, reported in [J. Biol. Chem. 271 (1996) 18651], cannot be mediated via increased [Ca2+]i.