A kinetic analysis of Na-Li countertransport in human red blood cells.

We examined the kinetic properties of the interactions between inner and outer cation sites of the Na-Li countertransport system in human red blood cells. Li-stimulated Na efflux [V(Na)] was measured as a function of external Li [(Li)o] and internal Na [(Na)i] contents. At each (Li)o, a Hanes plot of (Na)i/V(Na) vs. (Na)i allowed us to calculate the apparent dissociation constant for internal Na (KiNa) and the maximal rate of Na efflux [Vmax(Na)]. In erythrocytes from 10 different subjects, the Vmax(Na)/KiNa ratios were independent of the external Li concentrations. In other experiments, Na-stimulated Li efflux [V(Li)] was measured as a function of external Na and internal Li contents. In three subjects studied, the Vmax(Li)/KiLi ratios were independent of the external Na concentrations. The data strongly suggest that the countertransport mechanism is consecutive ("ping-pong").

Blood pressure and erythrocyte Na+ transport systems in a French urban male population.

This paper reports an investigation of blood pressure (taken as a continuous variable) as a function of: erythrocyte Na+ content; Na+,K+ pump; Na+,K+ cotransport and Na+,Li+ countertransport fluxes, and passive cation permeabilities in fresh erythrocytes from 129 French males who were living in an urban area and were not under treatment for any medical condition (after allowing for the effects of age, body mass index, alcohol and tobacco consumption). In contrast with previous findings in a North American population, we were unable to confirm that blood pressure was correlated with erythrocyte Na+ content and Na+,K+-AT-Pase activity. Conversely, the only transport parameter correlated (negatively) with blood pressure was outward Na+,K+ cotransport [r = -0.20, P less than 0.05 and r = -0.19, P less than 0.05, for systolic (SAP) and diastolic arterial pressure (DAP), respectively; n = 114]. When allowing for age, body mass index and alcohol consumption, the correlation coefficient between the Na+,K+ cotransport system and blood pressure increased from -0.20 to -0.28 (P less than 0.01) for SAP and from -0.19 to -0.28 (P less than 0.01) for DAP (n = 105). We conclude that the correlations between blood pressure and erythrocyte Na+ transport function could differ between North American and French (or Mediterranean) populations. In any case, a decreased pump or outward Na+,K+ cotransport activity may lead hypertensive subjects to a similar increase in cell Na+ (and Ca2+) content in the vascular wall.

[Na+, K+, Cl-]-cotransport function and dysfunction in different forms of primary hypertension.

We investigated the effect of an increase in cell Na+ content on outward and inward unidirectional fluxes catalyzed by the [Na+, K+, Cl-]-cotransport system in human erythrocytes (incubated in Li-Rb media). Erythrocytes with low Na+ content exhibited an uncoupled K+ efflux. The increase in cell Na+ content resulted in a more marked stimulation of outward Na+, K+ than of inward Li+, Rb+ cotransport fluxes (with stoichiometries not very different from one-to-one). These results suggest that in human erythrocytes and in nonepithelial cells with small but outwardly directed electrochemical Cl- gradients, the [Na+, K+, Cl-]-cotransport system may behave as a "second pump" by using the extra energy supplied by an additional net [K+, Cl-] efflux. The [Na+, K+, Cl-]-cotransport system (of vascular cells and/or noradrenergic endings) may play two different roles in primary hypertension: (a) "defective second pump" in some essential hypertensive patients with decreased cotransport affinity for internal Na+ and (b) "compensatory second pump" in other forms of primary hypertension where abnormalities in the Na+, K+ pump or in other ion transport systems may predispose the cell to a defective extrusion of excess cell Na+ content.

Inhibition of the Cl-/NaCO3- anion exchanger by xipamide in human red blood cells.

The mechanism of action of classical loop diuretics of the 2- or 3-amino-5-sulfamoylbenzoic acid and (aryloxy)acetic acid families involves competition with chloride for a common site on the (Na+, K+, 2Cl-) co-transport system. However this is not the mechanism of action of some high-ceiling diuretics like muzolimine, MK 473, xipamide, indapamide and clopamide, which are not carboxylic acids. We evaluated three of these latter diuretics (xipamide, muzolimine and clopamide) for their inhibitory effects on five ion transport systems in human red blood cells: (i) Cl(-)-dependent (Na+, K+) co-transport, (ii) (NaCO3-/Cl-) anion exchanger, (iii) (Cl-, K+) co-transport, (iv) Na+, K+ pump and (v) Na+: Li+ counter-transport; and on one ion channel the Ca2+-dependent, K+ channel. All erythrocyte transport pathways were resistant to the three diuretics studied (IC50 of 10(-3) M or higher) with one remarkable exception, the (NaCO3-/Cl-) anion exchanger. This transport system was inhibited by xipamide (IC50 of 2.5 +/- 0.4 X 10(-5) M, mean +/- S.D. of five experiments) and less potently by muzolimine (IC50 of 1.1 +/- 0.3 X 10(-4) M, mean +/- S.D. of three experiments). Clopamide only inhibited the anion exchanger at high concentrations (IC50 of about 10(-3) M). Xipamide, the most potent diuretic in this test, was at least one order of magnitude more active than furosemide, ethacrynic acid, hydrochlorothiazide and amiloride. Inhibition of the anion carrier could be involved in the diuretic action (inhibition of CO2-stimulated NaCl absorption in the TAL) and/or in the antihypertensive action (inhibition of net NaCO3- influx and secondarily of Ca2+ influx through Na+: Ca2+ exchange in vascular smooth muscle cells of xipamide).

The significance of the relative effects of loop diuretics and anti-brain edema agents on the Na+,K+,Cl- cotransport system and the Cl-/NaCO3- anion exchanger.

3-Amino-5-sulfamoylbenzoic acids and several series of (aryloxy)alkanoic acids were evaluated for their inhibitory effects on two human erythrocyte ion transport systems--the Na+,K+ cotransport system and the DIDS-sensitive anion carrier. Several classic loop diuretics, including the (aryloxy)alkanoic acid-ethacrynic acid and several 3-amino-5-sulfamoylbenzoic acids, like bumetanide and furosemide, displayed relatively strong inhibitory activity versus the cotransport system with relatively weaker action versus the anion carrier. Furthermore, diuretic potency correlated with cotransport inhibitory potency. Another class of (aryloxy)alkanoic acids, namely the [(2,3-dihydro-1H-inden-5-yl)oxy]acetic acids, such as indacrinone and MK-473, which exhibit less potent loop diuretic activity, were less potent cotransport inhibitors and more effective inhibitors of the anion carrier. Still other (aryloxy)alkanoic acids, with little saliuretic activity, namely a sub-class of [(2,3-dihydro-1H-inden-5-yl)oxy]alkanoic acids and a series of [(2,3,9,9a-tetrahydro-1H-fluoren-7-yl)oxy]acetic acids displayed little or no inhibitory action on the cotransport system but enhanced inhibitory action on the anion carrier. Most interestingly, the relative anion carrier inhibitory potency correlated well with the relative inhibitory activity of each compound on bicarbonate-stimulated cell swelling in cat cerebrocortical slices.

Erythrocyte passive potassium flux is increased in patients with ischemic coronary disease (ICD) and in subjects with family history of ICD.

BACKGROUND: It has been proposed that ischemic coronary disease (ICD) associated potassium loss could be due to modifications of potassium permeability. We investigated whether a positive family history of ICD can influence this parameter. We have compared potassium permeability in erythrocytes from ICD patients and from positive family history subjects (FICD) with control subjects. METHODS: All patients and subjects were carefully selected for the absence of hypertension and dysmetabolic pathologies. ICD group: 24 patients (19 males, 5 females; ages 43 to 69) all affected by ischemic coronary disease, under no drug treatment; FICD group: 18 subjects (all males, ages 27 to 42) with a verified positive ICD family history, without hypertensive family history and cardiovascular pathology; control group: 16 subjects (11 males, 5 females; ages 28 to 48) without positive family history of ICD. Passive potassium efflux (PPE) was spectrophotometrically measured in K-free medium containing ouabain and bumetanide. The kinetic constant was calculated by dividing PPE by the erythrocyte potassium concentration. RESULTS: No statistically significant differences were noted between the intracellular potassium content of the three groups. However, (1) the passive potassium permeability of the ICD group was significantly higher (kK=0.055 +/- 0.021 h(-1), n=24) than that of the control group (kK=0.023 +/- 0.008 h(-1), n= 16; p<0.00001), (2) the FICD group was higher (kK=0.036 +/- 0.012 h(-1), n=18) than the control group (p<0.001), and (3) the ICD group was higher than the FICD group (p<0.001). CONCLUSIONS: Our results suggest an inheritability of ICD, paralleling the familial aggregation of the pathology. Erythrocyte potassium permeability could represent an early marker of ischemic coronary disease and be used as a prophylactic tool.

Demonstration of a [K+,Cl-]-cotransport system in human red cells by its sensitivity to [(dihydroindenyl)oxy]alkanoic acids: regulation of cell swelling and distinction from the bumetanide-sensitive [Na+,K+,Cl-]-cotransport system.

A screening of several families of compounds on NEM-stimulated K+ efflux in human red cells allowed us to select a [(dihydroindenyl)oxy] alkanoic acid (DIOA) as the first potent inhibitor of this K+ flux (IC50 of 10(-5) M) without side effects on the bumetanide-sensitive [Na+,K+,Cl-]-cotransport system. Incubation of human red cells in hypotonic media (179 mosm) increased cell volume (by 18-20%) and provoked the appearance of a DIOA-sensitive K+ efflux of 4.48 +/- 0.83 mmol.(liter of cells X hr)-1 (mean +/- SD of nine experiments). This DIOA-sensitive K+ efflux exhibited a Michaelian-like dependence on the Cl- concentration of the incubation media (freely equilibrated with intracellular Cl-) with an apparent dissociation constant of 39.6 +/- 14.7 mM and a maximal rate of 4.7 +/- 0.9 mmol.(liter of cells X hr)-1 (mean +/- SD of five experiments). The chloride effect was mediated by intracellular and not by extracellular Cl-, as expected for an outward [K+,Cl-]-cotransport. The above properties of DIOA-sensitive K+ efflux clearly confirm that human red cells have a [K+,Cl-]-cotransport system that regulates cell swelling. The regulatory response to hypotonic media was also strongly depressed by cytochalasin B at a concentration of 1 mM, suggesting that the activating signal is probably transduced by the cytoskeleton.