L-Cysteine influx in type 2 diabetic erythrocytes

Erythrocyte oxidative stress has been implicated in the pathogenesis of diabetes mellitus, and the deficiency of antioxidant defense by the glutathione (GSH) pathway is thought to be one of the factors responsible for development of complications in diabetes. Erythrocytes require L-cysteine for the synthesis of GSH and the rate of synthesis is determined only by L-cysteine availability. In the present study we have found that the L-cysteine influx in erythrocytes from type 2 diabetic patients was significantly lower compared to age-matched controls. The decreased influx may be one of the factors leading to low GSH concentration observed in type 2 diabetes. Since L-cysteine is the limiting amino acid in GSH synthesis, any strategy aimed to increase L-cysteine influx in erythrocytes may be beneficial for type 2 diabetic patients.     

Investigation of the transport of intact glutathione in human and rat type II pneumocytes

The aim of the study was to investigate whether there is transmembrane transport of intact glutathione ([³H]-GSH, 0.1 μCi) in rat and human type II pneumocytes (T2P), and if this transport might be dependent on the redox state of the extracellular fluid. The T2P were pretreated with acivicin (250 μM) to inhibit γ-glutamyl-transferase activity and with L-buthionine-[SR]-sulfoximine (1 mM) to inhibit intracellular GSH synthesis. After 48 h in culture, initial GSH influx rate was 0.70 ± 0.20 nmol/min/mg protein (37°C) and 0.35 ± 0.04 nmol/min/mg protein (4°C) during the first 5 min in rat T2P. In human T2P, the initial GSH influx rate was 0.36 ± 0.30 nmol/min/mg protein (37°C) and 0.32 ± 0.06 nmol/min/mg protein (4°C) during the first 10 min. Thereafter no further influx was found. The influx of 1 mM GSH in freshly isolated rat and human T2P in suspension was 2.3 ± 0.3 and 1.2 ± 0.3 nmol/mg protein after 15 min at 37°C, and 2.8 ± 0.2 and 1.0 ± 0.3 nmol/mg protein at 4°C, respectively. When GSH influx was studied at different concentrations between 0 and 40 mM, a linear increase without saturation or difference between 37°C and 4°C was found. Preexposure to ouabain had no effect on GSH influx. Efflux of GSH was stimulated and influx inhibited by preexposure of the cells to reduced thiols, while disulphides inhibited efflux and favoured inward uptake. Thus, in human and rat T2P a GSH-carrier exists which operates as an effluxer. At GSH concentrations in the physiological range no uptake is seen, but some uptake can be observed at GSH concentrations above normal physiological levels. The uptake appears to be energy-independent and non-saturable. Efflux of GSH is stimulated and influx inhibited by reduced thiols, while disulphides inhibit the efflux and favour inward uptake. GSH uptake in T2P thus may depend on concentration gradients and driving forces, such as the redox state of the extracellular fluid.     

Investigation of the transport of intact glutathione in human and rat type II pneumocytes

The aim of the study was to investigate whether there is transmembrane transport of intact glutathione ([³H]-GSH, 0.1 μCi) in rat and human type II pneumocytes (T2P), and if this transport might be dependent on the redox state of the extracellular fluid. The T2P were pretreated with acivicin (250 μM) to inhibit γ-glutamyl-transferase activity and with L-buthionine-[SR]-sulfoximine (1 mM) to inhibit intracellular GSH synthesis. After 48 h in culture, initial GSH influx rate was 0.70 ± 0.20 nmol/min/mg protein (37°C) and 0.35 ± 0.04 nmol/min/mg protein (4°C) during the first 5 min in rat T2P. In human T2P, the initial GSH influx rate was 0.36 ± 0.30 nmol/min/mg protein (37°C) and 0.32 ± 0.06 nmol/min/mg protein (4°C) during the first 10 min. Thereafter no further influx was found. The influx of 1 mM GSH in freshly isolated rat and human T2P in suspension was 2.3 ± 0.3 and 1.2 ± 0.3 nmol/mg protein after 15 min at 37°C, and 2.8 ± 0.2 and 1.0 ± 0.3 nmol/mg protein at 4°C, respectively. When GSH influx was studied at different concentrations between 0 and 40 mM, a linear increase without saturation or difference between 37°C and 4°C was found. Preexposure to ouabain had no effect on GSH influx. Efflux of GSH was stimulated and influx inhibited by preexposure of the cells to reduced thiols, while disulphides inhibited efflux and favoured inward uptake. Thus, in human and rat T2P a GSH-carrier exists which operates as an effluxer. At GSH concentrations in the physiological range no uptake is seen, but some uptake can be observed at GSH concentrations above normal physiological levels. The uptake appears to be energy-independent and non-saturable. Efflux of GSH is stimulated and influx inhibited by reduced thiols, while disulphides inhibit the efflux and favour inward uptake. GSH uptake in T2P thus may depend on concentration gradients and driving forces, such as the redox state of the extracellular fluid.     

Bidirectional mechanism of plasma membrane transport of reduced glutathione in intact rat hepatocytes and membrane vesicles.

We determined the trans effects of extracellular reduced glutathione (GSH) on the rate of efflux of endogenous labeled GSH from freshly isolated rat hepatocytes. The presence of GSH (10 mM) in the medium significantly stimulated the fractional rate of efflux of [35S]GSH from 5.2 to 12.6%/15 min (p < 0.01). This effect was concentration-dependent, had sigmoid type of kinetics (D50 of 0.32 mM), and was reversible upon removal of external GSH. trans-Stimulation (counter-transport) was also observed with 5 mM oxidized glutathione (GSSG) and ophthalmic acid (fractional [35S] GSH efflux: 13.4% +/- 4.1 and 8.8% +/- 2.3 in 15 min, respectively, compared with control: 4.7 +/- 2.5/15 min). Bromosulphthalein-glutathione (BSP-GSH, 5 mM) in Krebs buffer inhibited the fractional [35S]GSH efflux (1.1%/15 min), whereas in Cl(-)-free buffer, GSH efflux was stimulated (14.2%/15 min) compared with control. trans-Stimulation was independent of chloride. BSP-GSH cis-inhibited and trans-stimulated the initial rate of GSH transport in basolateral-enriched membrane vesicles (bLPM) but not in canalicular-enriched membrane vesicles (cLPM). gamma-Glutamyl compounds also cis-inhibited and trans-stimulated GSH transport in bLPM vesicles. GSH-depleted hepatocytes incubated with 10 mM [35S]GSH accumulated more GSH than repleted cells, but the initial rate of uptake of radioactivity was faster in repleted cells. In contrast, repleted hepatocytes incubated with tracer or 50 microM [35S]GSH did not take up GSH. Thus, the sinusoidal membrane GSH transporter exhibits low affinity kinetics with sigmoid features for both GSH uptake and trans-stimulation of efflux, explaining the lack of uptake of GSH at low physiologic extracellular concentrations. Therefore, our findings support and explain the widely held view that GSH transport is unidirectional under physiologic conditions. However, the efflux of GSH may also occur in exchange for the uptake of organic anions and gamma-glutamyl compounds.     

High sulfhydryl content in turtle erythrocytes: is there a relation with resistance to hypoxia?

Glutathione concentration in the erythrocytes of the fresh-water turtle Phrynops hilarii, as determined by the enzymic recycling method is 1.9 +/- 0.2 mM. The erythrocyte non-protein -SH, NPSH, content, as determined by the 5,5' dithiobis(2-nitrobenzoic acid), DTNB, reducing capacity is 4.6 +/- 0.8 mM. The total DTNB reducing capacity in the erythrocytes, including hemoglobin, is 26 +/- 5 mM. Incubation with oxidized glutathione, greatly increases the electrophoretic mobility of both hemoglobin components present in the erythrocytes of Phrynops hilarii, indicating the formation of mixed disulphides with glutathione. The high --SH content in the erythrocytes of P. hilarii might be part of a redox buffering principally an antioxidant mechanism involved in resistance to hypoxia, possibly along the hypoxic period as well as during reperfusion with oxygen.     

Comparison of N-acetyl-L-cysteine and L-cysteine in respect to their transmembrane fluxes

The objective of the present study was to compare cysteine and N-acetyl-L-cysteine in respect to their transmembrane fluxes and find out which one is a better available precursor for the cells and thus better supports the intracellular glutathione synthesis. Cysteine can directly participate in glutathione synthesis, whereas N-acetyl-L-cysteine must be first deacetylated before its incorporation to glutathione. In the present study we investigated and compared the efficiencies of cysteine and N-acetyl-L-cysteine influx and efflux through the erythrocyte membrane. Erythrocytes transported both cysteine and N-acetyl-L-cysteine in a concentration-dependent manner. However, our results demonstrated that cysteine crosses the erythrocyte membranes more efficiently as compared to N-acetyl-L-cysteine. Treatment of erythrocytes with 5 mM of cysteine or N-acetyl-L-cysteine for 1 hr raised the intracellular free sulfhydryl group (free-SH) levels to 3.37 ± 0.006 or 2.23 ± 0.08 μ mol/ml erythrocyte, respectively. Cysteine more effectively than N-acetyl-L-cysteine restored the intracellular free-SH level depleted beforehand. In erythrocytes previously depleted of free-SH, 5 mM cysteine raised the free-SH level to 1.45 ± 0.075 μ mol/ml within 1 hr, whereas N-acetyl-L-cysteine at the same concentration raised this level to 0.377 ± 0.034 μmol/ml only. The results of our study also revealed that both cysteine and N-acetyl-L-cysteine influx and efflux processes are temperature dependent indicating that their transport requires biological activity. Our results demonstrate that cysteine is a better thiol precursor for the erythrocytes. Availability of cysteine for the cells is higher than that of N-acetyl-L-cysteine. The article is published in the original.     

Increased efflux of oxidized glutathione (GSSG) causes glutathione depletion and potentially diminishes antioxidant defense in sickle erythrocytes

Erythrocytes are both an important source and target of reactive oxygen species in sickle cell disease. Levels of glutathione, a major antioxidant, have been shown to be decreased in sickle erythrocytes and the mechanism leading to this deficiency is not known yet. Detoxification of reactive oxygen species involves the oxidation of reduced glutathione (GSH) into glutathione-disulfide (GSSG) which is actively transported out of erythrocyte. We questioned whether under oxidative conditions, GSSG efflux is increased in sickle erythrocytes. Erythrocytes of 18 homozygous sickle cell patients and 9 race-matched healthy controls were treated with 2,3-dimethoxy-l,4-naphthoquinone, which induces intracellular reactive oxygen species generation, to stimulate GSSG production. Intra- and extracellular concentrations of GSH and GSSG were measured at baseline and during 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. While comparable at baseline, intracellular and extracellular GSSG concentrations were significantly higher in sickle erythrocytes than in healthy erythrocyte after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation (69.9 ± 3.7 μmol/l vs. 40.6 ± 6.9 μmol/l and 25.8 ± 2.7 μmol/l vs. 13.6 ± 1.7 μmol/l respectively, P<0.002). In contrast to control erythrocytes, where GSH concentrations remained unchanged (176 ± 8.4 μmol/l vs. 163 ± 13.6 μmol/l, NS), GSH in sickle erythrocytes decreased significantly (from 167 ± 8.8 μmol/l to 111 ± 11.8 μmol/l, P<0.01) after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. Adding multidrug resistance-associated protein-1 inhibitor (MK571) to erythrocytes blocked GSSG efflux in both sickle and normal erythrocytes. GSSG efflux, mediated by multidrug resistance-associated protein-1, is increased in sickle erythrocytes, resulting in net loss of intracellular glutathione and possibly higher susceptibility to oxidative stress.     

Transport characteristics of system A in the rat exocrine pancreatic epithelium analyzed using the specific non-metabolized amino acid analogue alpha-methylaminoisobutyric acid

The selectivity and kinetics of system A amino acid transport in the rat exocrine pancreatic epithelium were characterized using the specific analogue alpha-methylaminoisobutyric acid. Unidirectional influx of alpha-methylaminoisobutyric acid was measured in isolated perfused pancreata by rapid dual tracer dilution. In cross-inhibition experiments DL-methylalanine, L-serine, L-cysteine, glycine, L-phenylalanine and L-glutamine were effective inhibitors of influx, whereas L-glutamate and L-lysine were less effective. In the presence of sodium alpha-methylaminoisobutyric acid influx was saturable with an apparent Kt = 1.7 +/- 0.2 mM and Vmax = 0.49 +/- 0.03 mumol/min per g (mean +/- S.E., n = 6). Influx of alpha-methylaminoisobutyric acid at 50 microM and 100 microM concentrations was significantly inhibited as the perfusate sodium concentration was gradually decreased from 156 mM to 26 mM by isoosmolar choline replacement. Estimated Kt values for sodium at these two methylaminoisobutyric acid concentrations approximated 200 mM. System A activity in the basolateral membrane of the exocrine pancreatic epithelium exhibits a high transport affinity, a wide tolerance for different amino acids and a dependency upon the extracellular sodium concentration.     

A one-to-one Mg2+:Mn2+ exchange in rat erythrocytes

Mg2+ efflux in rat erythrocytes was stimulated by increases in external Na+ concentration following a Michaelian-like function with an apparent dissociation constant (KNa) of 11 +/- 3 mM (mean +/- S.D. of three experiments) and a variable maximal rate ranging from 150 to 1200 mumol (liter (1) cells X h)-1. Na+-stimulated Mg2+ efflux was inhibited by quinidine and by ATP depletion. In the absence of external Na+, Mg2+ efflux was stimulated by increases in external Mn2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMn) of 35 +/- 15 microM (mean +/- S.D. of four experiments) and a variable maximal rate ranging from 350 to 1400 mumol (1 cells X h)-1. Mn2+-stimulated Mg2+ efflux was inhibited by quinidine, by ATP depletion, and by increasing the external Na+ concentration. Quinidine-sensitive (or ATP-dependent) Mg2+ efflux exhibited very similar values when compared with quinidine-sensitive (or ATP-dependent) Mn2+ influx. Mn2+ efflux in rat erythrocytes (loaded with total internal Mn2+ contents of 230-450 mumol/l cells) was stimulated by increases in external Na+ concentration and inhibited by quinidine. In the absence of external Na+, Mn2+ efflux was stimulated by increases in external Mg2+ concentration following a Michaelian-like function with an apparent dissociation constant (KMg) of about 35 +/- 5 microM (mean +/- range of two experiments) and a maximal rate of about 60-100 mumol (1 cells X h)-1. In conclusion, the Na+-stimulated Mg2+ carrier of rat erythrocytes may catalyze a one-to-one and reversible Mn2+:Mg2+ exchange in the absence of external Na+.     

Characterization of Na+/Mg2+ antiport by simultaneous 28Mg2+ influx

During net Mg2+ efflux from Mg2+-preloaded chicken erythrocytes, which occurs via Na+/Mg2+ antiport, 28Mg2+ is taken up intracellularly. Km of 28Mg2+ influx amounted to 1 mM. In Na+-free medium Vmax of 28Mg2+ influx was increased and Km was reduced to 0.2 mM. 28Mg2+ influx was noncompetitively inhibited by amiloride as was found for Na+/Mg2+ antiport. The results indicate that, extracellularly, Mg2+ can compete with Na+ for common binding sites of the Na+/Mg2+ antiporter, resulting in 28Mg2+-24Mg2+ exchange. The rate of Mg2+ exchange depends on extracellular Na+ and on the rate of net Mg2+ efflux.     

Analysis of glutathione-enhanced differentiation by microfilariae of Onchocerca lienalis (Filarioidea: Onchocercidae) in vitro

Reduced glutathione (GSH), but not its oxidized form (GSSG), stimulated development of Onchocerca lienalis microfilariae to the late first-larval stage in vitro. The degree and frequency of development was dose-related with a peak of activity at 15 mM, a concentration that is similar to known intracellular levels of GSH. To determine the mode(s) of action of this multifunctional compound, other reducing agents (L-cysteine, dithiothreitol), cysteine delivery agents (N-acetyl-L-cysteine, L-thiazolidine-4-carboxylic acid, L-2-oxothiazolidine-4-carboxylic acid), cysteine analogues (S-methyl-L-cysteine, D-glucose-L-cysteine, cysteine ethyl ester), free-component amino acids of GSH (glutamic acid, cysteine, and glycine), a specific metabolic inhibitor of gamma-glutamyl synthetase (buthionine sulfoximine), and an inhibitor of gamma-glutamyl transpeptidase (gamma-glutamyl glutamic acid) were also tested at concentrations of 0.01-50 mM in this system. N-acetyl-L-cysteine at 1-5 mM and D-glucose-L-cysteine at 2.5-10 mM significantly enhanced development. In contrast to those worms maintained in GSH-supplemented medium, microfilariae exposed to GSH for only the first 24 hr showed no enhancement by day 7 in culture. Neither buthionine sulfoximine nor gamma-glutamyl glutamic acid at 0.01-35 mM inhibited the effects of 15 mM GSH or 1 mM N-acetyl-L-cysteine. Results indicate that GSH or other cysteine analogues possessing a free sulfhydryl group must be present in the extranematodal environment to support microfilarial differentiation in vitro.     

Analysis of glutathione in rat airway surface liquid by capillary zone electrophoresis with conductivity detection

Glutathione (GSH) is an important component of antioxidant defenses in airway surface liquid (ASL), a thin layer (10-30 microm) of liquid covering the epithelial cells lining the airways of the lung. Decreased levels of ASL GSH have been reported in cystic fibrosis (CF), potentially contributing to the severe oxidative stress seen in this disease. To help investigate the role of GSH in ASL, we developed a technique suitable for analysis of GSH and its oxidized form (GSSG) in microliter samples using capillary sampling followed by capillary zone electrophoresis (CZE) analysis with conductivity detection. CZE was carried out in 100 mM CHES and 40 mM lithium hydroxide with 5 mM spermine at pH 9.1 under an applied electric field of -416 V cm(-1). To prevent any autooxidation of GSH during sample manipulations, the samples were treated with N-ethylmaleimide (50 mM) to alkylate free thiol (-SH). Under these conditions, GSH and GSSG were cleanly separated without interference from common anions (e.g. Cl(-), PO(4)(3-), HCO(3)(-), etc.) and the limit of detection for ASL analysis was 11 microM for GSH and 8 microM for GSSG (S/N=3). GSH and GSSG were also measured in rat plasma. Baseline values of 897+/-210 microM (GSH) and 215+/-61 microM (GSSG) were obtained for rat ASL (n=8), whereas 12.4+/-2.7 microM (GSH) and 14.8+/-6.7 microM (GSSG) were obtained for rat plasma (n=5).     

Calcium transport in intact human erthrocytes

Intact human erythrocytes can be readily loaded with calcium by incubation in hypersomotic media at alkaline pH. Erythrocyte calcium content increases from 15-20 to 120-150 nmol/g hemoglobin after incubation for 2 h at 20 degree C in a 400 mosmol/kg, pH 7.8 solution containing 100 mM sodium chloride, 90 mM tetramethylammonium chloride, 1 mM potassium chloride, and 10 mM calcium chloride. Calcium uptake is a time-dependent process that is associated with an augmented efflux of potassium. The ATP content in these cells remains at more than 60% of normal and is not affected by calcium. Calcium uptake is influenced by the cationic composition of the external media. The response to potassium is diphasic. With increasing potassium concentrations, the net accumulation of calcium initially increases, becoming maximal at 1 mM potassium, then diminishes, falling below basal levels at concentrations above 3 mM potassium. Ouabain inhibits the stimulatory effect of low concentrations of potassium. The inhibitory effects of higher concentrations of potassium are ouabain insensitive and independent of the external calcium concentration. Sodium also inhibits calcium uptake but this inhibition can be modified by altering the external concentration of calcium. The effux of calcium from loaded erythrocytes is not significantly altered by changes in osmolality, medium ion composition, or ouabain. It is concluded that hypertonicity increases the net uptake of calcium by increasing the influx of calcium and that some part of the sodium potassium transport system is involved in this influx process.     

The influx of calcium ions into human erythrocytes during cold storage

1. When human erythrocytes, suspended in iso-osmotic sucrose containing CaCl(2), are stored at 3 degrees C, Ca(2+) influx into the cells occurs. Simultaneously, efflux of K(+), Na(+), Cl(-) and water takes place and cell volume diminishes. 2. The extent of Ca(2+) influx increases with duration of cold storage and with increasing concentration of Ca(2+) in the suspending medium. 3. Erythrocytes that have been thus loaded with Ca(2+) exhibit Ca(2+) efflux against a concentration gradient when subsequently incubated at 37 degrees C. 4. Ca(2+) influx likewise occurs when the sucrose of the medium is replaced by iso-osmotic solutions of other non-ionized compounds. 5. Replacement of sucrose by iso-osmotic KCl or NaCl greatly diminishes the rate of Ca(2+) influx during cold storage; however, in iso-osmotic choline chloride, Ca(2+) influx is as rapid as in sucrose. 6. Preincubation of erythrocytes in iso-osmotic sucrose at 37 degrees C causes rapid efflux of K(+) and Na(+) and renders the cell membranes highly permeable to Ca(2+) during subsequent cold storage. 7. Preincubation of erythrocytes in iso-osmotic NaCl at 37 degrees C with trypsin or neuraminidase is without effect on the permeability of the membrane towards Ca(2+). 8. The experimental results lead to the conclusion that the main prerequisite for Ca(2+) influx into erythrocytes is the partial depletion of the cells of their univalent cations.     

Protective effects of glutathione and cysteine on the methylmercury-induced striatal dopamine release in vivo

The possible protective effects of glutathione (GSH), cysteine (CYS) and methionine (MET) on the Methylmercury (MeHg)-induced dopamine (DA) release from rat striatum were investigated using in vivo microdialysis coupled to HPLC with electrochemical detection. Intrastriatal infusion of MeHg 400 microM increased extracellular DA levels to 1941 +/- 199% in terms of basal levels. Infusion of MeHg 400 microM in GSH 400 microM pretreated animals, only increased striatal DA levels to 465 +/- 104%, in terms of basal levels, this increase being 76% lower than induced by MeHg alone. Conversely, the infusion of MeHg 400 microM after infusion of GSH 400 microM increased DA levels to 1019 +/- 96% in terms of basal levels, this increase being 47.5% lower than that observed in MeHg non-pretreated animals. The infusion of MeHg 400 microM in CYS 400 microM -pretreated animals, increased striatal DA levels to 740 +/- 149%, in terms of basal levels, this increase being 62% lower than that induced by MeHg in non-pretreated animals. The infusion of MeHg 400 microM in MET 400 microM pretreated animals increased striatal DA levels to 2011 +/- 230% in terms of basal, an increase that was not significantly different from that produced by MeHg 400 muM alone. In summary, the administration of compounds containing free -SH groups prevented the MeHg-induced DA release from rat striatum, probably due to the binding of MeHg to -SH groups. This would result in a lower metal availability to interact with -SH membrane proteins groups, which would decrease MeHg ability to interact with DA transporter.     

Volume-sensitive K transport in human erythrocytes

Studies have been carried out on human erythrocytes to examine the alterations of K transport induced by swelling or shrinking the cells by osmotic and isosmotic methods. Hypotonic swelling of erythrocytes (relative cell volume, 1.20) resulted in a striking, four- to fivefold augmentation in the ouabain-resistant K influx over the value obtained at a normal cell volume. Shrinking the cells in hypertonic media resulted in a small but statistically significant reduction in K influx. Three different methods of varying cell volume gave similar results. These include the addition of sucrose and of NaCl to hypotonic media and the isosmotic (nystatin) method. The major fraction of the K influx in swollen cells is specific in its requirement for Cl or Br and is not supported by thiocyanate, iodide, nitrate, methylsulfate, or acetate. Bumetanide (0.1 mM), MK-196 (0.2 mM), and piretanide (1 mM) are poorly effective in suppressing K uptake in swollen cells, but at higher concentrations, bumetanide (1 mM) inhibits 80% of the Cl-dependent K influx in swollen cells. The bumetanide concentration required to inhibit 50% of the Cl-dependent K influx is 0.17 mM. The volume-sensitive K influx is independent of both extracellular and intracellular Na, so that the (Na + K + 2Cl) cotransport pathway is not a likely mediator of the volume-sensitive K transport. A variety of inhibitors of the Ca-activated K channel are ineffective in suppressing swelling-induced K influx. Like K uptake, the efflux of K is also enhanced by cell swelling. Swelling-activated K efflux is Cl dependent, is independent of extracellular and intracellular Na, and is observed with both hypotonic and isosmotic methods of cell swelling. The activation of K efflux by cell swelling is observed in K-free media, which suggests that the volume-sensitive K transport pathway is capable of net K efflux. The addition of external K to hypotonic media resulted in an increase in K efflux compared with the efflux in K-free media, and this increase was probably due to K/K exchange. Thus, hypotonic or isosmotic swelling of human erythrocytes results in the activation of a ouabain-resistant, Cl-dependent, Na-independent transport pathway that is capable of mediating both net K efflux and K/K exchange.     

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer

The velocity of the oxidative renaturation of reduced ribonuclease A catalyzed by protein disulfide isomerase (PDI) is strongly dependent on the composition of a glutathione/glutathione disulfide redox buffer. As with the uncatalyzed, glutathione-mediated oxidative folding of ribonuclease, the steady-state velocity of the PDI-catalyzed reaction displays a distinct optimum with respect to both the glutathione (GSH) and glutathione disulfide (GSSG) concentrations. Optimum activity is observed at [GSH] = 1.0 mM and [GSSG] = 0.2 mM. The apparent kcat at saturating RNase concentration is 0.46 +/- 0.05 mumol of RNase renatured min-1 (mumol of PDI)-1 compared to the apparent first-order rate constant for the uncatalyzed reaction of 0.02 +/- 0.01 min-1. Changes in GSH and GSSG concentration have a similar effect on the rate of both the PDI-catalyzed and uncatalyzed reactions except under the more oxidizing conditions employed, where the catalytic effectiveness of PDI is diminished. The ratio of the velocity of the catalyzed reaction to that of the uncatalyzed reaction increases as the quantity [GSH]2/[GSSG] increases and approaches a constant, limiting value at [GSH]2/[GSSG] greater than 1 mM, suggesting that a reduced, dithiol form of PDI is required for optimum activity. As long as the glutathione redox buffer is sufficiently reducing to maintain PDI in an active form [( GSH]2/[GSSG] greater than 1 mM), the rate acceleration provided by PDI is reasonably constant, although the actual rate may vary by more than an order of magnitude. PDI exhibits half of the maximum rate acceleration at a [GSH]2/[GSSG] of 0.06 +/- 0.01 mM.     

Mechanisms of ascorbic acid recycling in human erythrocytes.

Vitamin C, or ascorbic acid, is efficiently recycled from its oxidized forms by human erythrocytes. In this work the dependence of this recycling on reduced glutathione (GSH) was evaluated with regard to activation of the pentose cycle and to changes in pyridine nucleotide concentrations. The two-electron-oxidized form of ascorbic acid, dehydroascorbic acid (DHA) was rapidly taken up by erythrocytes and reduced to ascorbate, which reached intracellular concentrations as high as 2 mM. In the absence of D-glucose, DHA caused dose-dependent decreases in erythrocyte GSH, NADPH, and NADH concentrations. In the presence of 5 mM D-glucose, GSH and NADH concentrations were maintained, but those of NADPH decreased. Reduction of extracellular ferricyanide by erythrocytes, which reflects intracellular ascorbate recycling, was also enhanced by D-glucose, and ferricyanide activated the pentose cycle. Diethylmaleate at concentrations up to 1 mM was found to specifically deplete erythrocyte GSH by 75-90% without causing oxidant stress in the cells. Such GSH-depleted erythrocytes showed parallel decreases in their ability to take up and reduce DHA to ascorbate, and to reduce extracellular ferricyanide. These results show that DHA reduction involves GSH-dependent activation of D-glucose metabolism in the pentose cycle, but that in the absence of D-glucose DHA reduction can also utilize NADH.     

Ca2+-activated Na+ fluxes in human red cells. Amiloride sensitivity

The effect of Ca2+ on the ouabain- and bumetanide-resistant Na+ fluxes in intact red cells was studied at relatively constant internal Ca2+, membrane potential, and cell volume. The red cell calcium concentration was modified using the ionophore A23187. In fresh red cells, the Na+ influx and efflux (1.2 +/- 0.13 and 0.26 +/- 0.07 mmol/liter cells x h, respectively) were not affected by amiloride (1 mM). When external Ca2+ was raised from 0 to 150 microM, in the presence of A23187, both the Na+ influx and efflux were stimulated (about 3.5-fold). The Ca2+-activated Na+ efflux and influx had an apparent Km for activation by Ca2+o of about 25 microM. The Ca2+-dependent Na+ transport was inhibited 30-60% by amiloride (ID50 = 17.3 +/- 8 microM). Amiloride, however, had no effect on the Ca2+-dependent K+ influx. The amiloride-sensitive (AS) transport pathway was a linear function of the Na+o concentration in the range from 0 to 75 mM. The Ca2+i activation seems to depend on the metabolic integrity of red cells. 1) It does not take place in ATP-depleted red cells; 2) ATP-repletion of ATP-depleted red cells fully restored AS Na influx; and 3) ATP-enrichment (ATP-red cells) enhanced the AS Na influx by about 100%. The Ca2+-activated AS Na+ influx was not affected by either DIDS or trifluoperazine. The present results indicate that in human erythrocytes an increase in internal Ca2+ activates on otherwise silent AS Na+-transport system, which is dependent on the metabolic integrity of the red cells.