In order to evaluate whether changes in plasma phospholipid composition are rapidly transmitted to the red blood cell membrane (RBCM) under in vivo conditions, the levels of major phospholipids in plasma, low density and high density lipoproteins (LDL and HDL) as well as in RBCM were determined before (pre), directly after (post) and 2 days after (48 h post) LDL apheresis in six patients with severe hypercholesterolaemia. LDL apheresis induced a 30-70% decrease in plasma and LDL cholesterol and total phospholipid levels within 23 h. Concomitantly, the percentages of plasma phosphatidylcholine (PC) and the PC/sphingomyelin (SM) ratio were increased compared to initial values. The percentage of plasma lyse PC (LPC) determined before apheresis in the patients was 30% lower with respect to the mean level of LPC in a normolipidaemic control. For LPC of LDL no differences were observed between normolipidaemia and hypercholesterolaemia. LDL apheresis induced a rise by about one third in the percentage of plasma LPC. At 48 h post, plasma LPC levels reapproached pre-apheresis levels, while the percentages of PC and the PC/SM ratio remained elevated. The pattern of changes induced by apheresis in plasma PC, SM and LPC levels was mimicked by changes in RBCM phospholipids. Strong positive relationships were noted for PC, SM and PC/SM as determined at pre, post and 48 h post between plasma and RBCM. In summary, changes in plasma PC, LPC, and PC/SM ratios as induced by LDL apheresis are rapidly transmitted to the RBCM under in vivo conditions, most probably as a result of phospholipid transfer between both compartments. In addition, the percentages of LPC in plasma and LDL particles of patients with severe hypercholesterolaemia are certainly below the levels of LPC reported to mediate biological effects of in vitro oxidized LDL.

In order to evaluate whether acute changes in fatty acids bound to phospholipids in plasma are transmitted into red blood cell membrane (RBCM) phospholipids, molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were analyzed after reduction of apo B containing lipoproteins through low density lipoprotein (LDL) apheresis in patients with severe hypercholesterolemia. As compared to the control, increases and decreases in molecular species with arachidonic acid (20:4) and with linoleic acid (18:2), respectively, at sn-2 of plasma diacyl-PC were seen in the patients before the apheresis. Directly after the procedure, the sum of species of plasma and RBCM PC plus PE with 20:4 were reduced. Two days after apheresis major species of plasma diacyl-PC reapproached preapheresis values while, in contrast, the composition of plasma alkenylacyl(plasmalogen)-PE was distinctly altered. In plasmalogen-PE of RBCM similar modifications were induced by the apheresis as in the same subgroup in plasma. In vitro experiments using vesicles with plasmalogen-PE labeled at sn-2 with either [14C]20:4 or a fluorescent pyrenedecanoyl residue indicated fast incorporation of the subgroup into the RBCM. In contrast, diacyl-PE was not taken up by the RBCM. In conclusion, apo B containing lipoproteins are partially responsible for the supply of phospholipids with arachidonic acid to RBCM, in particular by means of the fast incorporation of plasmalogen-PE. The transmission of changes induced by apheresis in plasma into those of the RBCM suggest that erythrocytes play an important role in the homeostasis of fatty acids bound to plasma phospholipids in vivo.

Among several phospholipid classes and molecular species of phosphatidylcholine and phosphatidylethanolamine (PE) analyzed, only the percentage of the molecular species 1-palmitoyl,2-arachidonoyl (16:0/20:4)-plasmalogen-(alkenylacyl)-PE showed positive relations to the maximal activity and to the dissociation constant of the red blood cell Na+/K+ pump for Na+ in normoand hyperlipidemic donors. A preferential interaction of this molecular species with the Na+/K+ pump is proposed.

Previous studies indicate a particular sensitivity of red blood cell Na+-Li+ countertransport activity to small variations in the fatty acid composition of membrane phospholipids. To assess whether the interindividual variability of Na+-Li+ countertransport is related to differences in the species pattern of erythrocyte phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vivo, the molecular species composition of PC and PE as well as the kinetics of Na+-Li+ countertransport were analyzed in parallel in normo- and hyperlipidemic donors. Both in diacyl PC and in diacyl-PE the species 16∶0/20∶4 and 16∶0/18∶2 were, respectively, positively and negatively related to the apparent maximal velocity of Na+-Li+ countertransport. The sum of all species with 20∶4 at sn2 of diacyl-PE exhibited a strong positive (r = 0.82, 2p < 0.001), and those containing 18∶2 a negative correlation (r = −0.63, 2p < 0.01) to the transport activity. Essentially similar connections were observed between these species and the apparent affinity of the transport system for intracellular Na+. To evaluate whether the associations between molecular species of membrane phospholipids and Na+-Li+ countertransport activity were indicative of a causal relationship, the species 16∶0/20∶4-PC and 16∶0/18∶2-PC were selectively introduced into the erythrocyte membrane by means of the PC-specific transfer protein. Replacement of 11% of native PC by 16∶0/18∶2-PC inhibited the transport rate by about 25%. Exchange of 6 and 9% of PC with 16∶0/20∶4-PC, in contrast, accelerated the transport rate by 30 and 60%, respectively. The accordance between the in vivo relations and the results of the in vitro modification strongly suggests that elevations and reductions in the arachidonic acid and linoleic acid content of membrane PC and PE contribute to the interindividual variability of red blood cell Na+-Li+ counter-transport activity and its acceleration in hyperlipidemias.

As compared to 7 normolipidemic donors, the maximal velocity of sodium-lithium countertransport was accelerated by nearly 70% in 10 patients with elevated levels of triglyceride-rich lipoproteins and tended to be stimulated also in 5 patients with hypercholesterolemia. No significant differences were observed between normolipidemia and both hyperlipidemic groups for the apparent affinities of the transport system for intracellular sodium and extracellular lithium. Strong positive relations of the maximal activity of sodium-lithium countertransport to the percentages of red cell membrane phosphatidylcholine (r = 0.85, 2P < 0.001), the phosphatidylcholine/sphingomyelin (r = 0.82, 2P < 0.001) and the phosphatidylcholine/phosphatidylethanolamine ratio (r = 0.81, 2P < 0.001) were seen in all donors. A negative correlation was found to membrane sphingomyelin (r = -0.72, 2P < 0.001). Also plasma phosphatidylcholine and sphingomyelin exhibited positive and negative associations, respectively, to the maximal activity of sodium-lithium countertransport (r = 0.66, 2P < 0.01 and r = -0.78, 2P < 0.001). Among several plasma lipoprotein parameters investigated, total triglycerides or VLDL cholesterol levels showed independent relations to both the plasma and the membrane phosphatidylcholine/sphingomyelin ratio as well as to the maximal velocity of sodium-lithium countertransport. The results indicate that an increase in red cell membrane phosphatidylcholine and a concomitant fall in sphingomyelin are closely associated with the acceleration of sodium-lithium countertransport in hyperlipidemia.

To examine the functional significance of epidermal growth factor (EGF) binding sites present on the human erythrocyte membrane [Engelmann et al. (1992) Am J Hematol 39:239–241], the effect of EGF on 45Ca2+ uptake and on 22Na+ efflux from these cells has been studied. In all cases media contained 1.25 mM Ca2+, whereas Na+ and K+ were varied. In 140 mM Na+/5 mM K+ medium EGF (250 ng/ml) stimulated 45Ca2+ uptake by 50%–90% in quin-2-loaded cells, and by up to threefold in untreated cells. Increasing extracellular K+ up to 75 mM at the expense of extracellular Na2+ stimulated the EGF-induced 45Ca2+ uptake by about twofold compared to 145 mM Na+ medium both in quin-2-loaded and in untreated cells. In 145 mM K+ medium, however, no EGF-induced 45Ca2+ uptake was detectable in quin-2-loaded cells, while in untreated cells Ca2+ entry was stimulated twofold by EGF. After increasing intracellular Na+ from 6 mmol/l cells to 18 mmol/l cells in untreated cells suspended in 145 mM K+ medium, 45Ca2+ uptake induced by EGF gradually increased. In contrast, in 140 mM Na+/5 mM K+ as well as in 70 mM Na+/75 mM K+ medium, 45Ca2+ uptake accelerated by EGF was largely unaffected by a modified red cell Na+ content. When 22Na-loaded untreated red cells were suspended in 145 mM K+ medium EGF stimulated red cell 22Na+ efflux by more than threefold. In 140 mM Na+/5 mM K+ as well as in 70 mM Na+/75 mM K+ medium, no 22Na+ efflux induced by the growth factor was evident. The results are consistent with the idea that EGF stimulates (at least) two components of 45Ca2+ uptake in human erythrocytes. One of the two is unmasked in 145 mM K+ medium, inhibited by quin-2 loading, accelerated by intracellular Na+ and appears to involve reversed Na+/Ca2+ exchange.

The molecular species composition of red blood cell diacyl-phosphatidylcholine (PC), diacyl-phosphatidylethanolamine (PE) and alkenylacyl-PE (plasmalogen PE) has been analyzed in normolipidemic and hyperlipidemic donors. In all three phospholipid subclasses the percentages of the species 16:0/20:4 were increased in hyperlipidemic patients. In diacyl-PE, 18:1/20:4 was also elevated. No changes were observed in the other quantitatively important molecular species containing arachidonic acid at sn-2, namely 18:0/20:4. The rise in 16:0/20:4 in diacyl-PC and diacyl-PE of hyperlipidemic donors was accompanied by a fall in molecular species with linoleic acid (18:2) at sn-2 (in particular 18:1/18:2). In alkenylacyl-PE the elevation of 16:0/20:4 was compensated by a decrease in species with docosatetraenoic acid (22:4) at sn-2 in particular by a fall in 16:0/22:4. Among all donors, the percentages of 16:0/20:4 in diacyl-PC and PE were positively associated with plasma total cholesterol levels. The changes in molecular species composition of PC and PE in hyperlipidemia are expected to alter the function of erythrocyte membrane transport proteins and--if present also in other cell types--to affect eicosanoid metabolism.

The composition of red blood cell membrane and plasma phospholipids has been analyzed in patients with hyperlipidemias. In red cells of patients with elevated levels of triacylglycerol-rich lipoproteins, phosphatidylcholine (PC) was raised and sphingomyelin (SM) reduced, resulting in a 20% increase of the membrane PC/SM ratio. In plasma phospholipids of these patients PC and SM levels were also higher and lower, respectively and the plasma PC/SM ratio was elevated by more than 50%. Close positive correlations between plasma and membrane phospholipids were obtained for PC, SM and the PC/SM ratio in normolipidemic and hyperlipidemic donors. Plasmalogen phosphatidylethanolamine (PE), a supposed endogenous protector against lipid oxidation, was reduced by about 20% in red cell membrane lipids in hyperlipidemic patients. Also plasmalogen-PE in plasma tended to be reduced in hyperlipidemic donors. Plasma HDL levels were positively related to the content of plasmalogen PE in the red cell membrane. In conclusion, there are closely related increases in PC/SM ratios in plasma and the red cell membrane in patients with elevated levels of triacylglycerol-rich lipoproteins. It is speculated that decreases in red cell membrane plasmalogen-PE in hyperlipidemic patients could be related to impaired antioxidant protection, possibly as a consequence of reductions in plasma HDL levels.

The effects of cholesterol loading and depletion and of a 10% replacement of native phosphatidylcholine by dipalmitoyl phosphatidylcholine (di 16:0-PC) on kinetic properties of human red cell Na−Li exchange have been studied.

Of eleven agglutinating lectins tested, only one, Ulex europaeus agglutinin I (UEA1), stimulated Ca2+ uptake in quin2-loaded erythrocytes by about 2-fold. UEA1 is known to be an α-l-focuse and ABH blood group specific lectin. The 45Ca2+ influx induced by UEA1 was absent in the presence of fucose (5 and 15 mM) and depended on the ABH blood group of the donor, the stimulatory potency of the lectin decreasing in the order H > A2 > A1. Ca2+ entry blockers, such as cobalt and verapamil, did not affect the 45Ca2+ influx induced by UEA1. 4,4′-Diisothiocyanatostilbene-2, 2′-disulfonic acid (DIDS) inhibited dose-dependently with a Ki of 1–2 μM. 10 μM DIDS, 10 μM 4,4′-dinitristilbene-2,2′-disulfonic acid (DNSD) and 20 μM dipyridamole fully blocked the 45Ca2+ influx induced by UEA1. The effect of UEA1 on 45Ca2+ influx was absent in K+ and Mg2+ media and was less pronounced in choline than in Na+ media. The 45Ca2+ influx induced by the lectin was abolished by preincubation with 12-O-tetradecanoylphorbol 13-acetate (TPA, 60 ng/ml). A monoclonal antibody raised against A1 erythrocytes (Bric 54) accelerated 45Ca2+ influx in quin2 loaded A1 erythrocytes b by about 2-fold. No effect was seen in A2 and H erythrocytes. The 45Ca2+ influx elicited by Bric 54 exhibited a sensitivity towards inhibition by DIDS and TPA, as well as a dependence on the cation composition of the incubation medium similar to that observed with UEA1. The effects of UEA1 and Bric 54 were not additive. These observations suggest that the Ca2+ influx induced by UEA1 and Bric 54 is mediated by the same transport pathway. Since both the lectin and the antibody exhibit ABH blood group specificity, it appears reasonable to conclude that ABH antigens can serve as recognition sites for activation of a Ca2+ influx pathway in human erythrocytes, which is sensitive to inhibitors of the band 3 anion-exchanger.

An assay was developed to characterize the kinetic parameters of the Na(+)-K+ pump of rat erythrocytes under conditions as physiological as possible. Changes in the red cell Na+ and Rb+ content were determined in Na+ media (containing 2.5 mM inorganic phosphate (PO4) as a function of cell Na+ (2-8 mmol/l) and extracellular Rb+ (0.2-5 mM). Evaluation of the data revealed that under these conditions the Na(+)-K+ pump mediates, in addition to forward running 3 Nai+: 2 Rbo+ exchange, 1 Ki+:Rbo+ exchange and pump reversal (3 Nao+:2 Ki+ exchange). The two latter modes of Na(+)-K+ pump operation are accelerated by PO4 and lowering of cell Na+. At physiological cation and PO4 concentrations, 1Ki+:Rbo+ exchange contributes by 30-60% to total ouabain-sensitive Rb+ uptake. Thereby, the stoichiometry of ouabain-sensitive Na+ net-extrusion to Rb+ uptake is reduced to values between 1.0 and 0.5. Only at cell Na+ contents above 20 mmol/l the Na+:Rb+ stoichiometry approaches the value of 3:2 = 1.5. At certain constellations of Nai+ and Rbo+ the Na(+)-K+ pump cannot perform any net-transport of Na+ and K+ (Rb+). These equilibrium points are not far from those expected from thermodynamic considerations. The results demonstrate that in normal rat erythrocytes the reversible reaction cycle of the Na(+)-K+ pump runs in several modes of operation. The "abnormal" modes complicate the interpretation of unidirectional fluxes mediated by the Na(+)-K+ pump.

Kinetics of ouabain-sensitive, furosemide-sensitive (FS), bumetanide-sensitive (BS) and -resistant Na+ and K+ transport were studied in erythrocytes of Prague hypertensive rats (PHR) and Prague normotensive rats (PNR). Maximal transport rates (Vmax) and apparent affinities for either intracellular Na+ or extracellular K+ (replaced by Rb+) were determined in red cells in which Na+ content varied around the physiological range and that were incubated in Na+ media. No major differences between PHR and PNR were disclosed in the kinetics of ion transport mediated by the Na(+)-K+ pump or BS inward Na(+)-K+ cotransport. FS Rb+ uptake was higher (due to a greater Vmax) in red cells of PHR as compared to PNR. In cells with a lowered Na+ content this elevation of FS Rb+ uptake was largely due to an augmented K(+)-Cl- cotransport which exhibits a low affinity for Rb+o and is blocked by 1 mM furosemide but not by 10 microM bumetanide. Red cells of PHR and PNR strains did not differ in either Na+ or Rb+ leaks. A slight increase of red cell Na+ content in PHR was evaluated in terms of the pump-leak concept. The present study did not reveal any obvious kinetic abnormalities of red cell cation transport the presence of which in tissues involved in blood pressure regulation would favor the development or the maintenance of genetic hypertension in PHR.

The uptake of chlortetracycline (CTC) and the nature of the fluorescence of CTC was studied in intact human erythrocytes from apparently healthy donors. The uptake of CTC at 22 degrees C proceeded with a t1/2 of about 3 min, and after 15 min a stable equilibrium was achieved with an intracellular accumulation by a factor of 5-6 relative to the medium concentration. The accumulation did not change in the range of CTC concentrations tested (20-500 microM). The Ca specificity of the CTC fluorescence spectrum was confirmed by Ca depletion of red cells using A23187 in the presence of EGTA and 0.2 mM Mg. This procedure decreased the total intracellular calcium content by about 70% and reduced the fluorescence intensity to one-fourth. Fluorescence microscopy of red cells incubated with 100 microM CTC at 22 degrees C showed that the fluorescence originated mainly from the red cell membrane. In addition, in about 15% of erythrocytes one or more fluorescent dots (diameter greater than 0.2 less than 1 microns) were detected. The fluorescence of the dots and membranes was related to calcium, as evidenced by the reduction of their intensity in Ca depleted cells. The number of erythrocytes with fluorescent dots and the frequency of the dots per cell was largely unaffected by lowering the incubation temperature to 0 degrees C, indicating that the dots most probably do not represent endocytotic artifacts induced by CTC. The number of dots was increased in erythrocytes preincubated with primaquine, demonstrating that CTC fluorescence can be applied to monitor the appearance of intracellular Ca storing vesicles. It is concluded that in (at least) 15% of erythrocytes obtained from apparently healthy donors intracellular vesicles containing Ca can be detected by CTC fluorescence microscopy.

1. Rates of erythrocyte Ca2+ inward transport, activities of erythrocyte Ca2(+)-adenosine triphosphatase, Na+, K+ pump, Na(+)-K+ co-transport and Na(+)-Li+ exchange, and erythrocyte Ca2+, K+ and Na+ contents have been determined in patients with carcinomas of the head and neck. 2. Decreases in Ca2+ inward transport and Na(+)-K+ co-transport activity, and increases in erythrocyte Ca2+ and K+ contents, were observed. 3. The more pronounced changes found in advanced carcinoma stages proved to be related to a 50% incidence of a mild anaemia of chronic disease in patients with carcinoma stages T3 and T4. 4. 'Young' (light) erythrocytes from healthy donors obtained by density centrifugation showed modifications in Na+ transport and K+ content similar to those seen in the patients. 5. It is concluded that the decrease in Na(+)-K+ co-transport activity, as well as the elevated K+ content and the lowered mean corpuscular haemoglobin content, but not the disturbances in Ca2+ homoeostasis, in erythrocytes of patients with carcinomas of the head and neck are most probably due to the occurrence of a mild anaemia of chronic disease in advanced carcinoma stages, associated with a reduction in mean erythrocyte age.

Blood pressure response to chronic high salt intake and kinetics of red blood cell Na+ and K+ (Rb+) transport were studied in salt-sensitive (DS) and salt-resistant (DR) Dahl rats fed a high salt diet (8% NaCl) for 7 weeks from the fifth (young), 12th (adult), or 23rd (old) week of age. The kinetics of ouabain-sensitive Rb+ uptake and Na+ extrusion were determined in Na+ media as a function of both intracellular Na+ (Na+i, 2-8 mmol/l cells) and extracellular Rb+ (Rb+o). In addition, the kinetics of furosemide-sensitive Rb+ uptake (related to Rb+o) and the magnitude of the Na+ and Rb+ leaks were assessed. High salt induced hypertension in young and adult but not in old DS rats although red blood cell Na+ was slightly increased in all age groups of DS rats fed a high salt diet. The kinetic parameters of the Na(+)-K+ pump were similar in DS and DR rats fed a low salt diet. Ouabain-sensitive transport rates were not suppressed in erythrocytes of salt hypertensive Dahl rats. Maximal velocities of the Na(+)-K+ pump (related to Na+i) decreased significantly with age in all groups except in DS rats fed a high salt diet. This was compensated by an age-dependent increase in the affinity for Na+i so that no substantial differences in transport rates between young and old rats were seen at physiological cell Na+ and plasma K+ levels.(ABSTRACT TRUNCATED AT 250 WORDS)

The kinetics of Na+ and K+ (Rb)+ transport mediated by the Na(+)-K+ pump and Na(+)-K+ cotransport system (assessed as a function of Rb+o and Na+i) as well as the magnitude of cation leaks were determined in red cells of young male rats subjected to chronic salt deprivation or salt loading (0.1% and 8% NaCl diet). These salt intake alterations induced moderate kinetic changes of the Na(+)-K+ pump which did not result in significant changes of ouabain-sensitive (OS) Rb+ uptake or Na+ net extrusion at in vivo Na+i and K+o concentrations because a decreased affinity for Na+i in salt-loaded animals was compensated by an increased maximal transport rate. High furosemide-sensitive (FS) Rb+ uptake in red cells of salt-deprived rats was caused by an increase of both the maximal transport rate and the affinity for Rb+o. Cation leaks were also higher in salt-deprived than in salt-loaded rats. In three age groups of rats fed a 1% NaCl diet FS Rb+ uptake (but not FS Na+ net uptake) rose with age due to an increasing maximal transport rate whereas the affinity of the cotransport system for Rbo+ did not change. The age-dependent changes in the kinetics of the Na(+)-K+ pump resulted in a slight decrease of OS Rb+ uptake with age that was not paralleled by corresponding Na+ net extrusion. No major age-related changes of cation leaks were found. Thus some intrinsic properties of red cell transport systems can be altered by salt intake and aging.

Erythrocyte Na+ and K+ transport mediated by the Na(+)-K+ pump, the Na+,K+ cotransport system and cation leaks, together with blood pressure, were determined in young and adult rats subjected to either chronic salt deprivation or chronic salt loading combined with subtotal nephrectomy. The kinetics of ion transport were studied in Na+ media as a function of extracellular K+, replaced by extracellular Rb+, and intracellular Na+ varied around the physiological range. A high salt intake increased blood pressure in young but not in adult subtotally nephrectomized rats. Erythrocyte Na+ or K+ contents of salt-deprived and salt-loaded rats did not differ. There were no major changes in Na+,K+ cotransport or cation leaks in salt-loaded rats. Chronic salt loading caused some alterations in the kinetics of the Na(+)-K+ pump, which were greater in young than in adult rats. The most pronounced change was a decreased affinity of the Na(+)-K+ pump for intracellular Na+, which was partially balanced by an increased maximal velocity. At physiological (in vivo) ion concentrations these kinetic alterations caused a slight reduction in total ouabain-sensitive Rb+ uptake [partly due to a decrease in intracellular K+:extracellular Rb+ (1:1) exchange] but no changes in Na+ net extrusion in salt-loaded rats. The erythrocyte Na+ and K+ transport systems showed no changes in intrinsic properties that would favour the development or maintenance of salt hypertension in young over adult rats if similar alterations occurred in tissues relevant for blood pressure control.

The nature of downhill Ca2+ net-transport into human erythrocytes was investigated using the experimental models of Ca2+ pump inhibition by vanadate and of intracellular chelation of Ca2+ by quin2. Ca2+ uptake by erythrocytes loaded with 0.5 mM vanadate and suspended in 145 mM Na+ -5 mM K+ media was reduced by about 60% when medium K+ was raised to 80 mM. Organic and inorganic Ca2+ entry blockers such as nifedipine (10(-5) M), verapamil (10(-4) M), diltiazem (10(-4) M), Co2+ (1.5 mM) and Cu2+ (0.1 mM) as well as the K+ channel blocker quinidine (1mM) inhibited Ca2+ uptake in 145 mM Na+ -5 mM K+ media by 60-75%. Flunarizine was less effective. In vanadate-loaded cells suspended in 70 mM Na+ -80 mM K+ media, in contrast, flunarizine exerted a dose-dependent inhibition of Ca2+ uptake by up to 80% at 10(-5) M, the other blockers being ineffective (except for verapamil at 10(-4) M). A similar pattern of inhibition was seen in quin2-loaded erythrocytes. The different susceptibility towards inhibitors may indicate that passive Ca2+ uptake by vanadate-loaded erythrocytes suspended in 145 mM Na+ -5 mM K+ media, on the one hand, and by vanadate-loaded erythrocytes suspended in 70 mM Na+ -80 mM K+ media as well as by quin2-loaded erythrocytes, on the other hand, is mediated by two different transport components.