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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.

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)

We described in this paper studies on the modes of operation of cuabain-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 subtrates 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 sodiumpotassium 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+.

Our previous observation of a greater increase in erythrocyte Na+ in SHR than in WKY after an acute Na+ load may result either from a genetic membrane property or from a specific plasma influence. In order to elucidate this question, membrane characteristics were compared with or without an acute Na+ load. Na+ transport was measured in Ringer and in plasma on Na+ enriched and K+ depleted red cells. Platelet microviscosity was measured as an index of membrane structural changes. After acute Na+ load a similar reduction of net Na+ extrusion and of K+ influx was observed in both strains. This indicates an inhibition of the Na+,K +-pump. Platelet microviscosity was similarly increased in SHR and WKY. Thus an acute Na+ load induced alterations of membrane properties in both SHR and WKY. The higher erythrocyte Na+ content in SHR stems rather from their intrinsic membrane properties than from a plasma factor.

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+.

Calcitonin is known to stimulate Ca2+ reabsorption and natriuresis and to increase adenosine 3',5'-cyclic monophosphate levels in early distal tubule, but its effects on acid-base transport mechanisms are not well characterized in this segment. We found that recovery of cell pH (pH1) from an induced acid load (using NH4+) in single isolated segments of the initial portion ("bright") of the rabbit distal convoluted tubule (DCTb) was due to an ethylisopropylamiloride-sensitive Na+/H+ exchanger both in the absence and presence of HCO3-, but we found no evidence for participation of other mechanisms such as an H+ pump or an HCO3(-)-dependent mechanism. Introduction of calcitonin stimulated an Na(+)-independent, HCO3(-)-dependent mechanism (0.17 +/- 0.04 pH units/min, n = 14) that reestablishes normal pHi after an induced acid load. This mechanism was observed only in the presence of CO2/HCO3- and was not inhibited by N-ethylmaleimide (1 mM), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (200 microM), or Sch-28080 nor stimulated by glutamine (2 mM) or ketoglutarate (0.5 mM), but it was dependent on chloride. We conclude that, in the DCTb, salmon calcitonin activates a latent Na(+)-independent, HCO3(-)-dependent mechanism.

Summary Canrenone inhibits 30–40% of ouabain-sensitive Na+ efflux in human red cells. Half-maximal inhibition was obtained with a canrenone concentration=86±37 μmol/l (mean±SD of 13 experiments). The partial inhibition of the Na+,K+ pump appears to be mediated at the digitalis receptor site with an apparent dissociation constant (KC)=200±130 μmol/l (mean±SD). Further evidence suggesting that canrenone is a partial agonist at the digitalis receptor site was obtained by the observation that it decreases the apparent affinity of the Na+,K+ pump for external K+. However, in contrast to ouabain, canrenone decreases the apparent pump affinity for internal Na+.

We present a model of steady state solute and water reabsorption along the rat proximal tubule. Major co-and counter-transport systems in the apical and basolateral cell membranes are described using kinetic descriptions based on data from the flows and solute concentrations along the length of the proximal tubule as a function of filtration rate and peritubular solute concentrations. We show that for many aspects of proximal tubule transport physiology this kinetics-based model is an adequate representation of the mammalian proximal tubule.

1. Erythrocyte cation transport pathways have been investigated in a family with pseudohyperkalaemia. 2. Ouabain- and bumetanide-resistant Na+ and K+ effluxes in three pseudohyperkalaemic patients were not different from those of control subjects when assessed at 37°C. 3. When the temperature was decreased to 20°C and 9°C, K+ passive permeability markedly increased and Na+ permeability remained unchanged in these patients. In contrast, in control subjects a reduction in temperature caused a marked reduction in Na+ and K+ passive permeability. 4. These findings could account for the marked increase in plasma K+ concentration observed at sub-physiological temperatures. 5. The Na+−K+ co-transport pathway was reduced in all members of the family, but the Na+−K+ pump was reduced in only two of them. These alterations were independent from the pseudohyperkalaemic state.

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+ co-transport 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.

1. In erythrocytes, the extrusion of a cell sodium load is accomplished by the ouabain-sensitive sodium-potassium pump and by the frusemide-sensitive sodium-potassium cotransport, which operate against the passive sodium permeability. All these three components of the cell sodium balance were studied in essential hypertension (410 subjects were investigated). 2. An abnormally low rate of net sodium extrusion by the sodium-potassium cotransport 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. 3. 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. 4. No difference could be detected between the passive sodium permeability of erythrocytes from hypertensive subjects and in those from normotensive controls. 5. In conclusion, essential hypertension seems to be associated with an inherited defect in 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.

Na+, K+-ATPase inhibitors extracted from plasma of healthy human subjects displaced3H-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.

Unidirectional22Na fluxes were investigated in human peripheral mononuclear leucocytes. Oua bain inhibited about 60% of Na efflux and the addition of bumetanide further reduced Na efflux rate by about 45%, sugesting the presence of a transport pathway capable of extruding Na against its gradient. Prostaglandins E1and E2and exogenous cAMP were found to be potent inhibitors of the bumetanide-sensitive Na efflux without affecting the ouabain-sensitive or the ouabain sensitive or the ouabain and bumetanide resistant Na effluxes.