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J Duhm

Publications and source records attributed to J Duhm.

At least 37 records · Page 2Linked to original sources
PubMed1989

Distinction of two components of passive Ca2+ transport into human erythrocytes by Ca2+ entry blockers.

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.

Aminoquinolines↗
PubMed1987

Physiology and biophysics of chloride and cation cotransport across cell membranes.

Many important questions remain to be answered about the mechanism that mediates coupled Na,K,Cl cotransport. We still do not know what the ATP requirement involves. Is ATP the direct energy source? Such an energy source does not seem to be necessary, inasmuch as the net free energy in the combined transmembrane chemical gradients of Na, K, and Cl is quite sufficient to maintain the observed high Cl(i). Could a protein kinase-mediated mechanism be responsible for the ATP requirement? How does reducing Cl(i) stimulate the transporter? What are the kinetic relationships for the co-ions at the outward- and inward-facing transport sites? Are they symmetrical? Can the squid axon regulate its cell volume? If so, is the Na,K,Cl transporter directly involved? Thus, the squid axon remains a fruitful preparation to study a transport mechanism similar to that found in a variety of cells. Its large size confers unique experimental advantages that should help us in our quest to understand this widely distributed transport mechanism.

Animals↗
PubMed1987

Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: modes of operation, dependence on extracellular and intracellular Na+, kinetics, pH dependency and the effect of cell volume and N-ethylmaleimide.

The effect of extracellular and intracellular Na+ (Nao+, Nai+) on ouabain-resistant, furosemide-sensitive (FS) Rb+ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na+ + 1 K+ + 2 Cl-) cotransport system operates in two different modes: mode i) promoting bidirectional 1:1 (Na+-K+) cotransport, and mode ii) a Nao+-independent 1:1 ki+ exchange requiring Nai+ which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb+ uptake through Rbo+/Ki+ exchange contributes about 25% to total Rb+ uptake in 145 mM NaCl media containing 5 mM RbCl at normal Nai+ (pH 7.4). Na+-K+ cotransport into the cells occurs largely additive to K+/K+ exchange. Inward Na+-Rb+ cotransport exhibits a substrate inhibition at high Rbo+. With increasing pH, the maximum rate of cotransport is accelerated at the expense of K+/K+ exchange (apparent pK close to pH 7.4). The apparent KmRbo+ of Na+-K+ cotransport is low (2 mM) and almost independent of pH, and high for K+/K+ exchange (10 to 15 mM), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na+ + 1 K+ + 2 Cl-) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987, Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl--dependent K+ transport pathway that is independent of both Nao+ and Nai+. This pathway differs in many properties from the basal, Nao+-independent K+/K+ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Nao+-independent FS K+ transport pathway which most probably is not identical to basal K+/K+ exchange. Ko+ less than Nao+ less than Lio+ less than Mgo2+ reduce furosemide-resistant Rb+ inward leakage relative to cholineo+.

Bumetanide↗
PubMed1987

Intracellular calcium content of human erythrocytes: relation to sodium transport systems.

To study the possible role of intracellular Ca (Cai) in controlling the activities of the Na+-K+ pump, the Na+-K+ cotransport and the Na+/Li+ exchange system of human erythrocytes, a method was developed to measure the amount of Ca embodied within the red cell. For complete removal of Ca associated with the outer aspect of the membrane, it proved to be essential to wash the cells in buffers containing less than 20 nM Ca. Ca was extracted by HClO4 in Teflon vessels boiled in acid to avoid Ca contaminations and quantitated by flameless atomic absorption. Cai of fresh human erythrocytes of apparently healthy donors ranged between 0.9 and 2.8 mumol/liter cells. The mean value found in females was significantly higher than in males. The interindividual different Ca contents remained constant over periods of more than one year. Sixty to 90% of Cai could be removed by incubation of the cells with A23187 and EGTA. The activities of the Na+-K+ pump, of Na+-K+ cotransport and Na+/Li+ exchange and the mean cellular hemoglobin content fell with rising Cai; the red cell Na+ and K+ contents rose with Cai. Ca depletion by A23187 plus EGTA as well as chelation of intracellular Ca2+ by quin-2 did not significantly enhance the transport rates. It is concluded that the large scatter of the values of Cai of normal human erythrocytes reported in the literature mainly results from a widely differing removal of Ca associated with the outer aspect of the membrane.

Antiporters↗
PubMed1987

Sodium transport across the red cell membrane and pathogenesis of essential hypertension: perspectives.

The possible roles of an acceleration of Na+-Li+ exchange and an alteration of Na+-K+ cotransport in the pathogenesis of hypertension are ascertained. Evidence is presented to indicate that the red cell Na+-Li+ exchange activity is goverened not only by genetic factors but also by plasma constituents that can vary in vivo. Of particular importance are the plasma K+ concentration and the plasma lipid composition. Red cell Na+-Li+ exchange activities increase with increasing saturation of the fatty acids in the membrane. An unifying hypothesis is proposed linking the primary change in plasma lipids to a secondary change of the lipid composition of cell membranes, resulting in alterations of lipo-protein interactions within the membrane and thereby in alterations of Na+ (and Ca2+) transport in several tissues, including erythrocytes. Such a mechanism could be responsible for development of high blood pressure in a subgroup of essential hypertensive patients that is characterized by a specific alteration in plasma lipid composition the precise nature of which remains to be investigated.

Animals↗
PubMed1986

Total intracellular calcium content and sodium transport in erythrocytes of essential hypertensive patients.

To estimate the amount of Ca bound to the inner aspect of the membrane of human erythrocytes, a method was developed to determine the total intracellular calcium content (Cai) using flameless atomic absorption. The mean Cai was 1.65 +/- 0.34 mumol/l cells in normotensive individuals (range 1.1-2.4). In untreated essential hypertensive patients Cai was slightly reduced, the difference being not significant. Antihypertensive treatment possibly affects Cai. Plasma ionized Ca and total plasma Ca tended to be lower and higher in the hypertensive patients, respectively. The activities of red cell Na-K co-transport, Na-Li exchange and the Na-K pump were inversely correlated to Cai in normotensives (P less than 0.05). The relations of Cai to Na-K co-transport and Na-Li exchange were displaced to higher values in untreated essential hypertensive patients.

Antiporters↗
PubMed1984

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytes in vitro and in vivo.

To study the physiological role of the bidirectionally operating, furosemide-sensitive Na+/K+ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145 mM NaCl media containing 0 to 10 mM K+ or Rb+. In pure Na+ media, furosemide accelerated cell Na+ gain and retarded cellular K+ loss. External K+ (5 mM) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb+ accelerated the Na+ gain like K+, but did not affect the K+ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na+/K+ transport system of human erythrocytes mediates an equimolar extrusion of Na+ and K+ in Na+ media (Na+/K+ "cotransport"), a 1:1 K+/K+ (K+/Rb+) and Na+/Na+ "exchange" progressively appearing upon increasing external K+ (Rb+) concentrations to 5 mM. The effect of furosemide (or external K+/Rb+) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na+ media, or with a cell swelling, indicating that the furosemide-sensitive Na+/K+ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5 mM external K+ or Rb+. The in vivo red cell K+ content was negatively correlated to the rate of furosemide-sensitive K+ (Rb+) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K+ and water-extruding mechanism under physiological conditions in vivo. The red cell Na+ content showed no correlation to the activity of the furosemide-sensitive transport system.

Biological Transport↗
PubMed1984

Na+-K+ transport and volume of rat erythrocytes under dietary K+ deficiency.

Red cell Na+ and K+ content and transport were studied in Sprague-Dawley rats in the course of a dietary K+ depletion ranging 1-6 wk. Plasma K+ fell to below 2 mM, and red cell K+ decreased. Cellular Na+ rose due to an increase of the Na+ leak. Inward Rb+ and outward Na+ transport by the Na+-K+ pump (determined at 2 mM external Rb+) were accelerated by the rise in cell Na+ concentration. K+ depletion caused a cation deficit of up to 30% of total red cell Na+ plus K+ and a consecutive cell shrinkage with an increase in mean cellular hemoglobin content (MCHC). The cell shrinkage, in turn, was paralleled by up to a 10-fold increase in the maximum capacity of the furosemide-sensitive, chloride-dependent Na+-K+ cotransport system. This system participated with up to 50% of the total K+ movements across the red cell membrane in severe K+ deficiency. In normal cells shrunken by osmotic means, Na+-K+ cotransport was similarly accelerated severalfold, indicating that the cell shrinkage occurring during K+ depletion is a major factor inducing the changes in Na+-K+ cotransport. However, a second unknown factor is also involved. It is concluded that in the rat, not only genetic but also environmental parameters contribute in determining the actual activity of the red cell Na+-K+ cotransport system. The cell volume and MCHC must be considered when judging Na+ and K+ transport changes observed in rat erythrocytes under various pathophysiological conditions.

Animals↗
PubMed1983

Platelet function, thromboxane formation and blood pressure control during supplementation of the Western diet with cod liver oil.

Epidemiologic and experimental data suggest an antiatherothrombotic potential of omega-3 polyunsaturated fatty acids. Therefore, the Western diet, which supplies predominantly omega-6 polyunsaturated fatty acids, was supplemented with 40 ml/day of cod liver oil, which provides about 10 g of omega-3 polyunsaturated fatty acids daily, for 25 days in eight volunteers. The omega-3 polyunsaturated fatty acids were incorporated in platelet and erythrocyte membrane phospholipids at the expense of omega-6 polyunsaturated fatty acids. Bleeding time increased (p less than 0.01) and platelet count (p less than 0.05), platelet aggregation upon ADP and collagen (p less than 0.01-0.05), and associated thromboxane B2 formation (p less than 0.01) decreased. Blood pressure (p less than 0.05) and blood pressure response to norepinephrine (p less than 0.01) and angiotensin II (NS) fell, without major changes in plasma catecholamines, renin, urinary aldosterone, kallikrein, prostaglandins E2 and F2 alpha and red cell cation fluxes. Biochemical and functional changes were reversed 4 weeks after cod liver oil was discontinued. Formation of prostaglandins derived from eicosapentaenoic acid and interference of eicosapentaenoic acid with formation and action of prostaglandins derived from arachidonic acid were evident in vitro. Whatever the mechanism, this moderate supplement of omega-3 polyunsaturated fatty acids markedly changed membrane phospholipids, which was associated with a shift toward less reactive platelets and a blunted circulatory response to pressure hormones.

Adult↗
PubMed1982

Transport pathways for lithium ions in neuroblastoma x glioma hybrid cells at 'therapeutic' concentrations of Li+.

The pathways of Li+ transport in neuroblastoma X glioma hybrid cells were studied at 2 mM external Li+. Five components of Li+ transport were identified. (1) A Na+-dependent Li+ countertransport system mediating Li+ transport in both directions across the plasma membrane. This transport pathway is insensitive to ouabain or external K+. It shows trans-stimulation (i.e. acceleration of Li+ extrusion by external Na+ and stimulation of Li+ uptake by internal Na+) and cis-inhibition (i.e. reduction of Li+ uptake by external Na+). (2) The Na+-K+ pump mediates Li+ uptake but not Li+ release in cells with physiological Na+ and K+ content. Li+ uptake by the pump in choline media is inhibited by both external Na+ and K+. In Na+ media, external K+ exhibits a biphasic effect: in concentrations up to about 1 mM, K+ accelerates, and at higher concentrations, K+ inhibits Li+ uptake by the pump. (3) Li+ can enter the voltage-dependent Na+ channel. Li+ uptake through this pathway is stimulated by veratridine and scorpion toxin, the stimulation being blocked by tetrodotoxin. Residual pathways comprise (4) a saturable component, which is comparable to basal Na+ uptake, and (5) a ouabain-resistant component promoting Li+ extrusion against an electrochemical gradient in choline media. The mechanisms for Li+ extrusion described here possibly explain how neuronal cells maintain the steady-state ratio of internal to external Li+ below 1 during chronic exposure to 1-2 mM external Li+.

Animals↗
PubMed1979

Studies on lithium transport across the red cell membrane. V. On the nature of the Na+-dependent Li+ countertransport system of mammalian erythrocytes.

Ouabain-resistant Na+-Li+ countertransport was studied on erythrocytes of man, sheep, rabbit, and beef. A transport system, exchanging Li+ for Na+ in a ratio of 1:1, was present in all four species. Li+ uptake by the exchange system increased 30-fold in the order man less than HK-sheep less than LK-sheep less than rabbit less than LK-beef. This order is identical to that of ouabain-resistant Na+-Na+ exchange in these species, but bears no relation to the Na+-K+ pump activity. The activity of the Na+-Li+ exchange system varied up to 7 and 16-fold among individual red cell specimens from man and beef, the variability being much smaller in sheep and rabbit erythrocytes. The affinities of the system for Li+ and Na+ were similar among the species and individuals (half saturation of the external site at about 1 mM Li+ and 50 mM Na+, respectively). 50-60% of Na+-Li+ exchange was blocked by N-ethylmaleimide in all species. p-Chloromercuribenzene sulfonate inhibited the exchange only in beef and sheep erythrocytes (60-80%). The two SH-reagents act by decreasing the maximum activity of the system, whilst leaving its affinity for Li+ unaltered. Phloretin was a potent inhibitor in all species. 1 mM each of furosemide, ethacrynic acid, and quinidine induced only a slight inhibition. The Na+-Li+ exchange of human and beef erythrocytes increased 3.5-fold upon elevation of the extracellular pH from 6 to 8.5, the pH-dependence arising from a change in affinity of the system for the cations and being similar to that reported for ouabain-resistant Na+-Na+ exchange in beef erythrocytes. It is concluded that a transport system exists in the red cell membranes of the four species which can mediate ouabain-resistant exchange of either Na+ for Na+, Na+ for Li+, or Li+ for Li+. The exchange system exhibits essentially identical transport characteristics in the four species, but shows a marked inter- and intra-species variability in maximum transport capacity and some differences in susceptibility towards inhibitors. A similar transport system is probably present also in other tissues. The exchange system seems to be distinct from the conventional Na+-K+ pump and shows no clear relation to one of the furosemide-sensitive, ouabain-resistant Na+ transport systems described in the literature.

4-Chloromercuribenzenesulfonate↗
PubMed1979

Studies on lithium transport across the red cell membrane. VI. Properties of a sulfhydryl group involved in ouabain-resistant Na+-Li+ (and Na+-Na+) exchange in human and bovine erythrocytes.

The reactivity of the SH-group essential for ouabain-resistant Na+-Li+ (and Na+-Na+) exchange and its location within the membrane are studied on human and beef erythrocytes and beef red cell ghosts. N-ethylmaleimide (NEM), 1,6-hexane dimaleimide, and iodoacetamide can induce an irreversible, partial inhibition of Na+-Li+ exchange in erythrocytes of the two species. The development of the inhibition due to the alkylating agents is greatly accelerated by external Na+ and Li+. The inhibition takes 3 min (NEM) and 60 min (iodoacetamide) to come to completion in isotonic Na+ media, but is hardly detectable in choline+, K+ or Mg2+ media. The transport site of the exchange system and the site promoting NEM binding exhibit similar affinities for external Na+. The impermeable, monofunctional glutathione derivative of 1,6-hexane dimaleimide does not inhibit Na+-Li+ exchange. The mercurials PCMBS, PCMB, and Hg2+ inhibit Na+-Li+ exchange in beef, but not in human erythrocytes. The inhibitory action of PCMBS, being slightly accelerated by external Na+, is fully reversed by penetrating thiols such as 2-mercaptoethanol, whilst glutathione, an impermeable thiol, is ineffective. Pretreatment with PCMBS affords partial protection from the irreversible inhibition caused by NEM. Oxidation with copper orthophenanthroline inhibits Na+-Li+ exchange only when performed in the presence of penetrating thiols such as 2-mercaptoethanol. It is concluded that the SH-reagents studied inhibit Na+-Li+ exchange by modifying an essential SH-group of a membrane protein in such a way that the turnover number of the exchange system is reduced. This SH-group is separated from both the red cell exterior and interior by a penetration barrier and seems to be distinct from the cation binding site. The action of external Na+ and Li+ in promoting the reaction of alkylating inhibitors is interpreted to result from a conformational change of the transport protein induced by the binding of external Na+ or Li+.

4-Chloromercuribenzenesulfonate↗
PubMed1978

Evidence for anionic cation transport of lithium, sodium and potassium across the human erythrocyte membrane induced by divalent anions.

1. The passive net transport of Li+ and Na+ across the human red cell membrane was accelerated by the divalent anions carbonate, sulphite, oxalate, phosphite and malonate. Phthalate, maleate, sulphate and succinate were found additionally to stimulate downhill transport of K+. Marked differences in anion efficacy and selectivity were observed. 2. The effects of these 'carbonate type' anions were reversible and fully blocked by SITS, dipyridamole and other inhibitors of anion transfer. 3. Cation transport acceleration induced by the monovalent anions salicylate, benzoate, thiocyanate and 2,4-dinitrophenol were inhibited by dipyridamole, but not affected by SITS. A great number of mono- and polyvalent anions were without detectable influence on Li+ transport. 4. Li+ net uptake induced by oxalate exhibited a pH dependence similar to that reported for halide self exchange. 5. Transport acceleration by carbonate type anions displayed a linear, 1:1 dependence on the concentrations of both the anion and the cation and was symmetric with respect to the two sides of the membrane. 6. It is concluded that the divalent carbonate type anions form singly charged, negative 1:1 ion pairs with the respective alkali metal cations, the ion pairs traversing the red cell membrane via the anion exchange pathway. This concept of anionic formation of some of the ion pairs considered. The relative efficacies and cation selectivities of polyvalent anions can largely be explained on the basis of electrostatic interactions governing ion pair formation. However, the chelating properties, structural flexibility, polarizability of the anions and the accessibility of the ion pairs to the anion exchange pathway need also be considered. 7. An exchange of NaCO-3 ion pairs for internal HCO-3 or Cl- is discussed as a possible mode of cellular pH regulation.

Anions↗