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Red blood cells are capable of transport and accumulation of catecholamines. The aim of this paper is to characterize the catecholamine transport system in the human red blood cell and in particular that of dopamine. Dopamine, noradrenaline and adrenaline enter the red blood cell by a similar process, which shows saturation kinetics with Vmax values of 0.54 +/- 0.12, 0.48 +/- 0.08 and 0.63 +/- 0.13 mumol (1 cells)-1 min-1, respectively, and K(m) values of 15.62 +/- 1.19, 5.81 +/- 1.19 and 12.00 +/- 2.97 nM, respectively. Observations based on the dependence of dopamine influx on the transmembrane H+ gradient, and the effect of transport inhibitors such as DMA (dimethyl-amiloride), DIDS (4,4'-diisothiocyanatostilbene 2,2'-disulphonic acid), reserpine, GBR 12909 (1-(2-(di(4-fluoro-phenyl)-methoxy)-ethyl)-4-(3-phenylpropyl)piperazine) , GBR 12935 (1-(2-(diphenyl-methoxy)-ethyl)-4-(3-phenyl-propyl)piperazine), and cyanine suggest that catecholamine transport is not mediated by the Na(+)-H+ exchanger, the anion exchanger or a system similar to that responsible for dopamine uptake in either synaptosomes or the proximal tubule. However, choline inhibits the influx of dopamine with an IC50 value of 17 microM and stimulates the efflux of dopamine with a K(m) value of 8.20 microM. These results strongly suggest that dopamine is transported by the choline exchanger previously reported to be present in red blood cells. Probenecid inhibits dopamine uptake with an IC50 of 0.63 microM. The presence of insulin receptors in human red blood cells, and the relationship between insulin and catecholamine levels in the plasma led us to investigate the effect of insulin on catecholamine transport. In fasting subjects, dopamine, adrenaline and noradrenaline influxes were higher than in fed subjects. Furthermore, the addition of exogenous insulin to red blood cells from fasting subjects significantly reduced the influx of catecholamines while no effect was observed when insulin was added to red blood cells obtained from fed subjects. The present study shows that catecholamines, and in particular dopamine, are transported in red blood cells via an exchanger which is possibly the choline transport system. The activity of this transporter is regulated by insulin. These results support a role for red blood cells as a storage pool for circulating catecholamines.

We have studied the kinetic properties of the interaction of internal Na with four different Na transport pathways (ouabain-sensitive Na, K pump, bumetanide-sensitive Na,K cotransport, Li:Na countertransport and passive Na permeability) in erythrocytes from 197 essential hypertensive patients. This study permitted us to classify 168 of the 197 hypertensives studied into the following groups: Pump - hypertensives-six patients showing low pump affinity for internal Na and high maximal pump rate; Co - hypertensives -95 patients characterized by low cotransport affinity for internal Na and low or normal maximal outward cotransport rate. Counter + hypertensives - 35 patients characterized by low countertransport affinity for internal Na and high maximal countertransport rate; and Leak + hypertensives - 32 patients showing increased passive Na permeability. Having studied the apparent affinity of the Na,K cotransport system for external K we divided the co - hypertensives into three subgroups (symmetric, asymmetric or antisymmetric) according to the presence of low, normal or high K affinity. Forty-five hypertensive patients were submitted to antihypertensive treatment with a new diuretic and antihypertensive drug, cicletanide. Preliminary results indicate that one of the factors involved in the antihypertensive efficiency of the compound is the subgroup to which the hypertensive belongs.

The Na+ and K+ electrochemical gradients across cell membranes are believed to be maintained by the action of a Na+, K+-pump. In human erythrocytes this pump exchanges internal Na+ for external K+ in approximately a 1.5 ratio. Thus, when Na+-loaded/K+-depleted erythrocytes are incubated in physiological conditions they tend to recover their original low Na+/high K+ content. Surprisingly, in erythrocytes from healthy donors the net Na+ extrusion/K+ influx ratio exceeds the 1.5 ratio predicted for Na+, K+-pump-mediated fluxes whereas it is similar to this value in erythrocytes from essential hypertensive patients and some of their descendants. We now report that this difference is due to the presence of a Na+, K+-co-transport system in normal erythrocytes which extrudes both internal Na+ and K+ and is functionally deficient in erythrocytes of essential hypertensive patients and some of their descendants. No difference in passive Na+ permeability could be detected between normotoensive and hypertensive subjects.

Pseudo-hyperkaliemia is a biochemical abnormality suspected when the blood level of K is increased, contrasting with the absence of usual symptoms of hyperkaliemia. The diagnosis is confirmed by the discrepancy between K+ blood levels which are normal if measured immediately after the blood sample is drawn, and increased if the blood sample is incubated at room temperature. In our case, the pseudo-hyperkaliemia is due to the passage of K outside the erythrocytes through increased passive membrane permeability to K. In vivo, this increased passive outflow of K seems to be compensated by an increased active inflow of K dependent on the sodium pump. This explains the absence of true hyperkaliemia and clinical symptoms. In vitro, it seems that the sodium pump is no longer able to ensure this increased activity. Therefore, the abnormality becomes overt, mirrored by a delayed increase in kaliemia.

Cyclic AMP inhibits the bumetanide-sensitive Na+,K+ cotransport system in human red cells. The cotransport inhibition is enhanced by addition of phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine to the incubation medium. The cyclic AMP concentration giving half-maximal cotransport inhibition showed a wide variation among different individuals (from 0.1 to 5 mM external cyclic AMP concentration). In contrast to cyclic AMP, cyclic GMP showed little effect on the cotransport system. The ouabain-sensitive Na+,K+ pump was almost unaffected by cyclic nucleotides. Prostacyclin was the only tested prostaglandin showing an effect on Na+ and K+ transport in human red cells. In some individuals, this icosanoid stimulated the Na+,K+ cotransport system. Leukotriene B4 stimulated K+ fluxes.

Plasma Na+, erythrocyte Na+ content and the activity of Na+ transport systems of red cells were measured in Wistar rat fed a normal or high Na+ diet. Net Na+ and K+ fluxes of erythrocytes were also measured in the presence of plasma of rats fed with excess Na+. Na+-K+ cotransport and passive Na+ permeability were increased. Erythrocyte Na+ content was increased after 2 months but not after 8 days of high Na+ intake. No significant difference in plasma Na+ and pump activity could be detected after such a diet. No factor acting in Na+ extrusion was found to be present in plasma of salt loaded rats. These results indicate that Na+ intake may modulate Na+ transport systems, namely passive permeability and Na+-K+ cotransport and that increased Na+ erythrocyte content is not a causative factor.

Na+,K+-ATPase inhibitors extracted from plasma of healthy human subjects displaced 3H-ouabain binding to human erythrocytes and inhibited the Na+ efflux catalyzed by the Na+,K+-pump and unexpectedly the Na+,K+-cotransport system without alteration of the Na+,Na+-exchange or the Na+ passive permeability. This suggests the presence in healthy human plasma of endogenous factors with ouabain-like and furosemide-like activities.

Different Na transport pathways were studied in the erythrocytes of 10 normotensive subjects who received 240 meq/day of Na in excess of their usual diet. In most of these subjects the maximal rate (Vmax) of the Na,K pump and the Na,K-cotransport system was markedly decreased on the first day of the diet. In some of these subjects, excess Na intake induced an increase in the apparent affinity for internal Na for the Na,K pump and the Na,K-cotransport system. The decrease in the Na,K pump fluxes was not concomitant to that of the co-transport system and not accompanied with an increase in blood pressure or cation concentration in the plasma. Interestingly, the apparent affinity for internal Li of the Li-Na exchange was markedly increased without alteration of the Vmax. The passive permeability for Na and the cellular Na content were not altered by excess Na intake. Ouabain and bumetanide at low concentrations respectively induced an increase in the apparent affinity for internal Na of the Na,K pump and the Na,K- cotransport system. These results are similar to those observed after excess Na intake. These later agree with the hypothesis that Na homeostasis regulates some endogenous factors with ouabain-like and furosemide-like properties that might contribute to the regulation of cellular Na handling.

Outward Na+ - K+ cotransport in erythrocytes from essential hypertensive Caucasian subjects was found to be excessively low (Co -) compared to normotensives (Co +) carefully selected for their negative family history of hypertension. Since the frequency of essential hypertension varies widely among different populations and is particularly high in certain coloured peoples, we compared erythrocyte Na+ - K+ cotransport in normotensive and hypertensive subjects in Paris (France) and in Abidjan (Ivory Coast) to seen whether defective cotransport was related to high blood pressure in the African group as well. Of the 66 French unselected normotensives investigated, 26 (39%) were Co - whereas 14 of the 18 Ivory Coast unselected normotensives (79%) were Co -. 64 (80%) of the 80 essential hypertensives examined in France were Co -, but the proportion of Co - subjects among the Ivory Coast hypertensives was even higher. In addition, both hypertensives and normotensives in the African groups often had undetectable outward Na+ effluxes, a rare finding in the French subjects. We suggest that the high incidence of abnormal Na+ - K+ cotransport in the Ivory Coast series may reflect a genetic propensity to hypertension in this population, and that consequently, Na+ - K+ erythrocyte cotransport measurements might prove useful in defining geographic variations in hypertension.

The red cells from 5 related patients with hereditary stomatocytosis were investigated. Maximal rate constant of Na+ passive permeability was increased while that of K+ passive permeability was nearly normal. Ouabain-sensitive Na+ efflux was elevated. The Na+ component of furosemide-sensitive Na+, K+ cotransport was also increased. However, its K+ component, determined in 2 patients, remained within normal limits, thus departing from the strict 1:1 stoichiometry of the Na+, K+ cotransport system. Yet, intracellular Na+ and K+ concentrations displayed limited and inconstant changes. A variety of abnormally-shaped cells, including stomatocytes, were observed in scanning electron micrographs. Upon differential centrifugation, reticulocytes usually concentrated in the most dense region of the gradient. Red cell deformability, as studied by ektacytometry, was reduced. Membrane phosphatidylcholines and sphingomyelins were increased and decreased, respectively, where-as fatty acid distribution was unchanged. Membrane microviscosity was normal.

Net fluxes of sodium and potassium ions were determined in sodium-loaded, potassium-depleted erythrocytes from 370 white subjects, 194 of whom had essential hypertension or had been born to parents with essential hypertension. Findings were compared with those in 86 controls who were normotensive and did not have a family history of hypertension. Compared with controls all patients with essential hypertension had a low sodium to potassium ratio secondary to a deficit in the sodium-potassium cotransport system. A similar abnormality was found in subjects born to parents with essential hypertension, the prevalences of a deficient cotransport system in such subjects being 53.6% (52 out of 97) among those with one hypertensive parent and 73.7% (14 out of 19) among those with two hypertensive parents. Both sexes were equally affected. Studies in 14 families over two or three generations showed the erythrocyte cation abnormality in one or more members of each consecutive generation. No close association was evident between the deficient erythrocyte sodium-potassium cotransport system and either blood groups ABO, Rh, Kidd, Duffy, P, and MNS or the major histocompatibility HLA antigens. Out of 90 consecutive unrelated and normotensive white blood donors, 36 showed a low erythrocyte sodium-potassium net flux ratio. It is concluded that in white people abnormal erythrocyte cation transport is a biochemical disorder characteristic of essential hypertension and transmitted by a dominant and autosomal mode expressing a single abnormal gene.

We describe in this paper studies on the modes of operation of ouabain-insensitive sodium transport systems in red cells of normotensive and hypertensive patients. We have extensively investigated the properties of Na countertransport and cotransport in order to clarify whether they are two different proteins or one transport protein with two modes of operation. Several criteria of discrimination between the two pathways are described: They differ in their affinity for Na and Li, sensitivity to several inhibitors, changes in cell volume, and chloride replacement by nitrate. We propose that there are two different transport systems. We have found elevated countertransport in red cells of hypertensive patients in France and in the United States. However, the cotransport system was found elevated in patients in Boston but reduced in patients in Paris. Studies of the modes of operation of the Na-K cotransport system indicate that it can promote K accumulation using an inward sodium gradient. This mode might be more efficient than Na extrusion at the physiological level of Na and K gradients. We interpret our findings of elevated Na-K cotransport in American hypertensive patients as an increased number of transport units functioning as K accumulators. It remains to be determined whether the reduced affinity for internal sodium of the outward cotransport is a defective outward cotransport or else a modulation of this transport system to favor K accumulation.

In erythrocytes, the extrusion of a cell sodium load is accomplished by the ouabain-sensitive sodium-potassium pump and by the furosemide-sensitive sodium-potassium cotransport, which operate against the passive sodium permeability. The precise characterization of these transport pathways requires the determination of the turnover rates of cation translocation and the affinities for substrates and effectors. The preliminary results of such kinetic study in essential hypertension is reported here. An abnormally low rate of net sodium extrusion by the sodium-potassium co-transport system was observed in essential hypertensive patients and in a high proportion of their young normotensive offspring. A normal cotransport system found in secondary hypertensive subjects devoid of familial history of hypertension confirmed that the abnormal cotransport system is not the consequence of high blood pressure per se. At the molecular level, the cotransport abnormality seems to be consecutive to a diminished apparent affinity for intracellular Na+. A 20-40% increase in the rate of net sodium extrusion by the sodium-potassium pump seems to compensate for the abnormal cotransport in erythrocytes from some young normotensive subjects born of essential hypertensive parents and from some benign essential hypertensive subjects. No difference could be detected between the passive sodium permeability of erythrocytes from hypertensive subjects and normotensive controls. In conclusion, essential hypertension seems to be associate with an inherited defect in the apparent affinity for intracellular Na+ of the sodium-potassium cotransport system. We propose therefore the laboratory study of this system for (i) the distinction between essential and secondary hypertension and (ii) the preventive investigation of young normotensive subjects in hypertensive families.

In recent studies were reported an inherited membrane defect which is closely related to the development of essential hypertension. This abnormality consists of a functional deficiency in the Na+--K+ co-transport mechanism in erythrocytes of essential hypertensives and some normotensives born of hypertensive parents. Here we report a case of juvenile essential hypertension including familial erythrocyte Na+, K+ measurements. The 2-year-old boy had essential hypertension and bore the abnormality, with no compensatory activity of the Na+, K+ pump. The mother had developed hypertension during pregnancy. She also bore the erythrocyte abnormality as the net Na+/K+ flux ratio was reduced. One sister bore the same abnormality without hypertension but a complete analysis of Na+ extrusion mechanisms in this patient demonstrated a compensatory effect of the Na+, K+ pump activity. Other members of the family were normotensive without the flux abnormality. It appears therefore that erythrocyte flux measurements might be of diagnostic and genetic interest in juvenile hypertension.

The kinetic parameters of the Ca2+ pump were assessed in red blood cells of essential hypertensive subjects as compared to their respective controls. Uphill Ca2+ efflux was investigated in Ca2+ -saturated intact red blood cells using a new method recently developed for human red cells (Dagher,G. and Lew, V. J. Physiol. (London), in the press). 45Ca-equilibrated cells were obtained using ionophore A23187 and Ca2+ efflux was assessed after addition of excess CoCl2 which totally inhibits Ca2+ influx and thus exposes uphill Ca2+ extrusion by the pump. The results comprise methodological aspects of the use of this technique in rat red blood cells. The determination of the maximal velocity and the Ca2+ concentration for half-maximal stimulation (KCa 0.5) did not reveal any alteration in essential hypertensives and spontaneously hypertensive rats as compared to their controls.

Outward Na+, K+-cotransport was 50% higher in female than in male rat red cells (50%). Ovariectomy induced a marked decrease in the activity of this system. Oestradiol increased and progesterone decreased Na+, K+-cotransport in ovariectomized rats.

1. The clinical applications of a simple laboratory test for the diagnosis of essential hypertension were further investigated on a large number of patients (124 individuals). 2. The reduced erythrocyte ratio of Na+/K+ net fluxes observed in essential hypertensives might be secondary to a functional defect in a Na+-K+ cotransport system. 3. A similar reduction in the ratio of Na+/K+ net fluxes was also observed in erythrocytes of some normotensive subjects born of hypertensive parents and of genetically hypertensive rats. This suggests that the erythrocyte membrane defect follows the genetic transmission of hypertension.

In erythrocytes from essential hypertensive patients and some of their normotensive offspring, the Na+,K+ co-transport system is unable to transport Na+ in an uphill direction against the electrochemical Na+ gradient. Recent studies of the kinetic properties of this system have permitted us to further characterize the abnormal co-transport in hypertension. Thirty-four essential hypertensive patients were characterized by a two- to three-fold decrease in the maximal rate of the outward Na+,K+ co-transport and a partial uncoupling of this system as compared with 22 normotensive controls. On the other hand, a normal co-transport was found in five secondary hypertensives with no family history of hypertension, thus indicating that the abnormal co-transport is not the consequence of high blood pressure per se. An abnormal co-transport was also found in around 50% of 19 young normotensives born of one hypertensive parent, suggesting genetic transmission. This study allows us to propose the use of a specific Na+,K+ co-transport assay for the differential diagnosis between essential and secondary hypertension and for the detection of those normotensives born of hypertensive parents with a potential risk to develop high blood pressure.

1. The uphill calcium efflux through calcium-saturated pumps in intact red cells was investigated with the aid of a new method, in initial conditions of uniform ionophore A23187-induced calcium distribution among the cells. The method is based on findings by Tiffert, García-Sancho & Lew (1984) which show that cobalt can suddenly arrest passive calcium transport by the ionophore and expose, without noticeable interference, uphill calcium extrusion by the pump. The results comprise methodological aspects and questions concerning interactions between inner pump sites, ATP and Ca2+, and the calcium: ATP stoichiometry of the calcium-saturated pump. 2. Ionophore-induced calcium influx was set to be far in excess of the maximal calcium pump capacity. This secured a uniform calcium distribution among the cells, and Ca2+ equilibration by 2 min or less of calcium permeabilization. Cobalt was added between 15 s and 5 min after ionophore addition. The calcium and ATP content of the cells was followed during ionophore-induced influx and cobalt-exposed efflux. 3. The external cobalt concentrations required to block completely ionophore-mediated calcium transport were similar or only marginally higher than those of calcium. 4. The reproducibility of independent cobalt-exposed calcium efflux measurements from single blood samples was within an 8% range. 5. During cobalt-exposed calcium efflux, the calcium content of subpopulations of cells, with and without active Ca2+-sensitive K+ channels, investigated by post-incubation of samples in low-K+, thiocyanate (SCN-) media (modified from García-Sancho & Lew, 1988a), was similar. This is consistent with the maintenance of a uniform calcium distribution among the cells during uphill calcium extrusion. 6. Cobalt-exposed calcium efflux was similar in the interval from 15 s to 5 min after calcium permeabilization although cell ATP levels had fallen by over 50% in that period. Therefore, cell ATP concentrations within the physiological range do not seem to be regulatory for calcium-saturated pumps in the intact red cell. 7. All cobalt-exposed calcium efflux curves were linear in time, at least until total cell calcium contents reached levels below 100 mumol/l cells. This suggests that internal calcium is not inhibitory for calcium-saturated efflux in intact cells in the 0.1-1 mmol/l cells range. 8. The cobalt-exposed calcium fluxes were in the range from 4 to 24 mmol/(1 cells.h) for fresh cells and from 10 to 18 mmol/1 cells. h) for samples from the Blood Bank.(ABSTRACT TRUNCATED AT 400 WORDS)

An abnormally low sodium-potassium net flux ratio in erythrocytes was recently described in human essential hypertension. We have confirmed this finding in 65 patients with essential hypertension who were compared with 33 normotensive controls born of normotensive parents. In 23 other subjects with documented secondary hypertension and normotensive parents, the sodium-potassium net flux ratio was found to be similar to that in the controls. The erythrocyte test thus appears to be of interest in distinguishing between essential and secondary hypertension. Severe renal failure itself reduces the flux ratio and would therefore distort the results of this test.

External K+ inhibits the maximal rate of outward Na+, K+ cotransport in human red cells with no effect on the apparent affinity for internal Na+. The K+ concentration giving half-maximal inhibition (KIK) varied from 16 to 30 mM in 24 normotensive control subjects. Six of the 38 hypertensive patients showed a KIK above the upper limit of this normal range. Only three hypertensive patients showed a KIK below normal range. The internal Na+ content giving half-maximal stimulation of outward Na+, K+ cotransport (KSNa) was measured in the hypertensive patients (a normal range of KSNa = 9 to 16 mmol/liter cells was previously established in 50 normotensive control subjects). Eighteen hypertensive patients showed an abnormally high KSNa, as previously described in hypertensive patients whose Na+, K+ cotransport system had a low affinity for internal Na+ (Co -). Comparison of KSNa with KIK showed that all six hypertensive patients with high KIK and all three hypertensive patients with low KIK were Co - hypertensive.

We report in this paper the effect of metabolic depletion on several modes of furosemide-sensitive (FS) Na and K transport in human red blood cells. The reduction of ATP content below 100 mumol/liter cells produced a marked decrease in the maximal activation (Vmax) of the outward, FS transport of Na and K into choline medium in the presence of ouabain (0.1 mM) and 1 mM MgCl2. The K0.5 for internal Na to activate the FS Na efflux was not altered by metabolic depletion. However, metabolic depletion markedly decreased the Ki for external K (Ko) to inhibit the FS Na efflux into choline medium (from 25 to 11 mM). Repletion of ATP content by incubation of cells in a substrate-rich medium recovered control levels of Vmax of the FS Na and K fluxes and of Ki for external K to inhibit FS Na efflux. The Vmax of FS Na and K influxes was also markedly decreased when the ATP content dropped below 100 mumol/liter cells. This was mainly due to a decrease in the inward-coupled transport of K and Na (NaO-stimulated K influx and the Ko-stimulated Na influx). The FS Ki/Ko exchange pathway of the Na-K cotransport, estimated from the FS K influx from choline-20 mM Ko medium into cells containing 22 mmol Na/liter cells, was also reduced by starvation. Starvation did not inhibit the FS Nai/Nao exchange pathway, estimated as FS Na influx from a medium containing 130 mM NaCl into cells containing 22 mmol Na/liter cells. The unidirectional FS 22Na efflux and influx were also measured in control and starved cells containing 22 mmol Na/liter cells, incubated in a Na medium (130 mM) at varying external K (0 to 20 mM). In substrate-fed cells, incubated in the absence of external K, FS Na efflux was larger than Na influx. This FS net Na extrusion (400 to 500 mumol/liter cells X hr) decreased when external K was increased, approaching zero around 15 mM Ko. In starved cells the net Na extrusion was markedly decreased and it approached zero at lower Ko than in substrate-fed cells. Our results indicate that the FS Na and K fluxes, and their major component, the gradient driven Na-K-Cl cotransport system, are dependent on the metabolic integrity of the cells.

Different Li, Na and K transport pathways were assessed in erythrocytes from manic-depressive patients. No alteration in the Li-Na countertransport, Na,K cotransport or passive permeabilities was observed in either unipolar or bipolar manic-depressive patients. In addition, acute or chronic lithium treatment did not alter the maximal velocity of either the Li-Na countertransport or the Na,K cotransport. A two-fold reduction of the ouabain-sensitive Na efflux was observed among manic-depressive patients without alteration in the affinity of the Na pump for internal Na.

Na, K-ATPase inhibitors extracted from plasma of healthy human subjects displacedH-ouabain binding to human erythrocytes and inhibited the Na efflux catalyzed by the Na, K-pump and unexpectedly the Na, K-cotransport system without alteration of the Na, Na-exchange or the Na passive permeability. This suggests the presence in healthy human plasma of endogenous factors with ouabain-like and furosemide-like activities. [ABSTRACT FROM AUTHOR]