Human red blood cells from prenatal hemopoiesis. Lithium flux (sodium dependent) asymmetry

In red cells from umbilical cord blood it has been referred the existence of lithium fluxes (contralateral sodium dependent) asymmetry. On account of the relevancy of this transport system in some pathologies it is pertinent the study of its kinetics to relate normal with pathological states in which it is affected. Lithium fluxes--contralateral sodium dependent were determined in N-ethylmaleimide treated neonatal red blood cells. Experimental data were fitted by simple Michaelis-Menten kinetics, finding Km and Vmax variables. It was shown the persistency of asymmetry. The independence of sulfhydryl groups (or the occultation of the groups involved to this inhibitor) could explain asymmetry persistence.(AU)

Human red blood cells from prenatal hemopoiesis. Lithium flux (sodium dependent) asymmetry

In red cells from umbilical cord blood it has been referred the existence of lithium fluxes (contralateral sodium dependent) asymmetry. On account of the relevancy of this transport system in some pathologies it is pertinent the study of its kinetics to relate normal with pathological states in which it is affected. Lithium fluxes--contralateral sodium dependent were determined in N-ethylmaleimide treated neonatal red blood cells. Experimental data were fitted by simple Michaelis-Menten kinetics, finding Km and Vmax variables. It was shown the persistency of asymmetry. The independence of sulfhydryl groups (or the occultation of the groups involved to this inhibitor) could explain asymmetry persistence.

Neonatal red blood cells: amiloride-insensitive Na+-H+ transport isoform would express Na+-Li+ exchange.

Neonatal red cells (umbilical cord blood) were in vitro incubated in isotonic media (thiocyanate as predominant anion). This experimental condition was selected as it was known that this chaotropic anion induced activation of Na+-H+ exchange. The transport amiloride-sensitive mechanism, that decreased the acidic intracellular pH change occurring in this medium, would correspond to Na+-H+ exchange (NHE1 isoform). However, the Na+-Li+ exchange, also determined in the cells in the above-mentioned medium was not affected by amiloride. The present data suggest that an amiloride insensitive Na+-H+ exchange isoform would express Na+-Li+ countertransport in these cells.

Na+/Li+ exchange kinetic characterization. Red blood cells from normotensive individuals.

The plasma membrane Na+/Li+ exchange is of research interest because it is involved in some pathologic states. A kinetic analysis of this transport system would be of greater importance as compared to the flux determination under "standard" conditions (reactants saturating levels) as a way to define normal values. Unidirectional lithium fluxes in red blood cells from normal adults were determined as a function of "trans" (contralateral) sodium ion concentration. Relating Li+ efflux and influx (Na+ "trans") kinetic parameters we found free carrier reorientation between the two phases (asymmetry).

Red blood cells Li+-Na+ exchange kinetics. Normal adolescents with hypertensive ancestors.

Li/Na exchange kinetic parameters (Km, Vmax) were determined in red blood cells from normal adolescents with or without hypertensive antecessors, as well as plasmatic levels of high density lipoproteins. In red blood cells from adolescents with hypertensive antecessors, we observed significantly lower Km values compared to the other group. HDL-c (high density lipoprotein-cholesterol) plasmatic values did not differ significantly. Furthermore, in normal adolescents without hypertensive ancestors, the Vmax significantly correlated with the HDL-c plasmatic values. The results indicated that the Km parameter of this transport mechanism is affected by genetic factors and data are also presented to demonstrate that the Vmax parameter is affected by environmental (cellular) factors. It is suggested that Li/Na exchange Km values would be a risk index of essential hypertension development.

Human red blood cells from prenatal hemopoiesis. Lithium flux (sodium dependent) asymmetry.

In red cells from umbilical cord blood it has been referred the existence of lithium fluxes (contralateral sodium dependent) asymmetry. On account of the relevancy of this transport system in some pathologies it is pertinent the study of its kinetics to relate normal with pathological states in which it is affected. Lithium fluxes--contralateral sodium dependent were determined in N-ethylmaleimide treated neonatal red blood cells. Experimental data were fitted by simple Michaelis-Menten kinetics, finding Km and Vmax variables. It was shown the persistency of asymmetry. The independence of sulfhydryl groups (or the occultation of the groups involved to this inhibitor) could explain asymmetry persistence.

Human red blood cells from prenatal hemopoiesis. Lithium flux (sodium dependent) asymmetry.

In red cells from umbilical cord blood it has been referred the existence of lithium fluxes (contralateral sodium dependent) asymmetry. On account of the relevancy of this transport system in some pathologies it is pertinent the study of its kinetics to relate normal with pathological states in which it is affected. Lithium fluxes--contralateral sodium dependent were determined in N-ethylmaleimide treated neonatal red blood cells. Experimental data were fitted by simple Michaelis-Menten kinetics, finding Km and Vmax variables. It was shown the persistency of asymmetry. The independence of sulfhydryl groups (or the occultation of the groups involved to this inhibitor) could explain asymmetry persistence.

Human red cells from prenatal hemopoiesis. Sodium/lithium exchange symmetry.

Human red blood cells from prenatal stages of hemopoiesis (umbilical cord blood) and with increased lithium content were submitted to media with varying sodium concentrations in order to characterize extracellular sodium dependent-lithium efflux. On the other hand cells with different sodium contents were submitted to lithium media in order to characterize cellular sodium dependent-lithium influx through the above system. Experimental data were fitted by simple Michaelis-Menten kinetics. The Km value for lithium efflux (extracellular sodium-dependent) was significantly lower than the Km value for lithium influx (cellular sodium -dependent). The Vmax value for lithium influx was significantly greater than that for lithium efflux. The Vmax/Km ratio values were independent from the way in which the fluxes were measured. It is concluded that the simple carrier cannot be excluded as a model of the Na+-Li+ exchange system in these cells.

Human red cells from pre (hepato splenic-late fetal) and postnatal (bone marrow-adult's) stages of haemopoiesis: Na+/Li+ exchange kinetic.

The kinetic parameters of Na(+)-Li+ exchange were studied in both neonatal and adult's red blood cells in order to evaluate if the asymmetry for Li+ fluxes (Na(+)-contralateral-dependent) is expressed in both types of cells. Maximum velocities (Vmax) and Km (half-activation constant) were measured for Li+ fluxes in both types of cells. In human neonatal red blood cells (nRBC), extracellular Na+-dependent Li+ efflux was shown to be a saturable function of external sodium concentration with high affinity (apparent Km: 2.1 mM) and low capacity (maximal velocity Vmax = 0.3 mmoles Li+ 1('''10 cells h(-1)). The Vmax and apparent Km for cellular Na(+)-dependent Li+ influx were higher (Km = 12.9 mM; Vmax = 1.07 mmoles Li+ 1(-1) cells h(-1)). The results provide evidence for intrinsic functional asymmetry in this transport system, as the transporter is more prevalent and stable in the inward-facing conformation. These kinetic observations may be explained in terms of the simple carrier transport model. Similar findings were found for this system in human adult's red cells. These results point to the asymmetry pattern (related to Na+ activation of Li+ flux) as developed in red cells from late prenatal stages of hemopoiesis.

Lead effects on structural and functional cellular parameters in human red cells from a prenatal hematopoiesis stage.

An attenuation (or reversion) of the prolytic effect of lead on neonatal red cells is observed in iso- or hypotonic low ionic strength media. This effect correlates neither with concomitant activation of K+ (Ca2+ or Pb2+) channels nor with volume reduction. Neonatal erythrocytes were used in this study owing to their greater cellular density, as compared with adult red cells, for the above mentioned channels. The attenuation-reversion effect would be mediated through lead interactions with the cytoskeleton, a structure that is the limiting factor for red cell lysis in low ionic strength media.

Human red cells from prenatal stages of hemopoiesis. Lithium flux components.

Red cells from umbilical cord with increased lithium content were submitted to different experimental conditions in order to study lithium flux components. There appeared three components: First, an ouabain-sensitive component, related to Na+ replacement with Li+ in the primary active Na+/K+ transport system. The magnitude of this fraction is greater than in adults' red cells. Second, an outside sodium-dependent Li+ efflux fraction, corresponding to the Li+/Na+ countertransport system with Vmax and K(m) values of 0.1 (mmol/l cells.h) and 2.58 (mmol/l), respectively. The Na+o-affinity for lithium efflux in this system is greater in neonatal than in adults' red cells. Third, a leak fraction with an equal value to that reported in adults' red cells. Furthermore, the possible non-existence of a bumetanide-sensitive lithium flux fraction was shown in neonatal red cells.

Calcium ionophore (A23187) differential effect on red cells from pre and postnatal haemopoiesis.

Differences in ionic transport mechanisms through human red cell membranes from pre and postnatal haemopoietic stages of development have been related. The analysis of calcium-sensitive conductive potassium pathways [K+ (Ca2+)] was realized from intracellular calcium level increase after treatment of cells with ionophores. The effect of calcium ionophore (A23187) on potassium transport in human neonatal red cells (nRC) was studied and compared with red cells from adults (aRC). Analysis focused on intra/extracellular potassium redistribution and potassium conductance [K+ (Ca2+)] values in incubated red cells suspensions. A difference was observed between neonatal and adult's red cells only for the first of the parameters referred to. Conductance values of the same order of magnitude for these potassium transport pathways between both cell types suggest that the results presented could rest on different [K+ (Ca2+)] cellular density.

The homogeneous effect of calcium ionophore A23187 on potassium loss in human foetal red cell populations.

A "pulse like" increase of cytoplasmic calcium concentration, which is proportional to ionophore concentration, is induced in red cells by exposure to A23187. Different Ca2+ levels are attained depending on cellular calcium buffering power and/or primary active calcium transport activation. We examined the effect of A23187 concentration of potassium loss in neonatal (nRC) as well as in adult red cells (aRC). The increase in ionophore concentration produced an "all- or -none" recruitment in adult cells and a "gradual" one in neonatal red cells. The "gradual" response observed in nRC would suggest that the "all or none" character of the response is not present in red cells during the foetal stages of haematopoiesis.

Unconjugated bilirubin effect on 3H-ouabain binding to human fetal red cells.

Human fetal red cells show heterogeneity of 3H-ouabain binding sites. These cells were chosen as a model to look into unconjugated bilirubin effects on the primary active Na(+)-K+ transport mechanism. Evidences are presented suggesting that unconjugated bilirubin affects 3H-ouabain binding but not through a direct effect. This is supported by the fact that the "low affinity" subgroup sites of the last mentioned ligand persists after unconjugated bilirubin treatment of cells, whereas the "high-affinity" subgroup disappears.

Human neonatal red blood cells. An experimental model for the study of calcium-activated potassium channel refractoriness.

To study some characteristics of calcium-activated potassium channels free from plasma membrane lipid perturbations, neonatal red blood cells were selected. To this end, cells not previously treated (ATP depletion and/or reversible lysis) were exposed to the ionophore A23187 in a NO3 medium with calcium. The net efflux of potassium from the cells was studied. Preincubation in a medium devoid of potassium induced a significant decrease only in the rate constant of potassium flux. The data suggest that, in the absence of plasma membrane lipid impairment, changes in internal sites reactivity with calcium ions of channel proteins would be involved in the refractoriness. This would be at variance with channel density modifications.

Neonatal red blood cell lysis induced by hypertonic low ionic strength media.

Human neonatal red cells (placental blood) incubated in hypertonic sucrose media showed a significative lytic process in a relatively short time interval. The addition of sodium chloride into the sucrose media reduced the extent of hemolysis. In contrast, the addition of calcium chloride enhanced the hemolysis in these red cells. Calcium-membrane components complex formation that destabilize membrane's bilayer structure would explain the calcium effect above mentioned (on account of the low ionic strength media used and exposed fixed negative charges) This study intends to clarify, in neonatal red cells, the relation between surface charges and cellular stability.

Human neonatal red cells. Regulatory volume response under anisotonic conditions.

Studies have been carried out in human neonatal red cells (cord blood) to examine the outcome of cellular volume under anisotonic media. When exposed to hypertonicity these cells showed, after an initial shrinkage phase, a volume expansion that reached short of the original volume at 60 minutes. The chloride replacement by nitrate as well as amiloride (in chloride media) inhibited this regulatory volume response. The cellular sodium increase that paralleled the volume expansion was also amiloride-sensitive. We suggest that a Na+/H+ antiport mechanism could mediate the response. Cell volume increase in hypotonic media was not followed by a shrinkage phase.

Neonatal red blood cells as a model for the study of ouabain-furosemide interaction on active K+ transport. A theoretical approach.

In a former paper by Serrani et al. (1990), an overlapping action of ouabain and furosemide on potassium influx was observed. In the present paper we demonstrated that Na, K-ATPase from neonatal red cells ghosts is a target of both drugs. Furthermore the combined action of ouabain and furosemide on potassium (86Rb) influx (in its greatest fraction mediated by primary active transport) was studied by generalized equations for the analysis of inhibitors. The isobologram and the combination index performed from the data obtained point to the existence of synergism or antagonism between both inhibitors according to the fraction affected. Clinical implications of these findings could be postulated.

Potassium influx in human neonatal red blood cells. Partition into its major components.

The potassium influx in human neonatal red blood cells (nRBC) shows an approximately 25% lower value compared to the total potassium influx in adult red blood cells (aRBC). The ouabain-sensitive potassium influx component represents approximately 70-75% of the total potassium influx for both types of cells but with an absolute value significantly lower in nRBC. In nRBC, the half maximum inhibitory effect for ouabain was obtained at a 10(-9) M concentration. The ouabain-insensitive nRBC potassium influx fractions showed two components: (i) a bumetanide-sensitive component, significantly lower than that of aRBC, (ii) a ouabain-bumetanide-insensitive (leak) component with a similar value in both cell types. The sum of the ouabain-sensitive and furosemide-sensitive components amounted in nRBC to a greater value than the total potassium influx. This behaviour could be interpreted as a superposition of the action of the inhibitors on the components affected.

States of stability/lysis in human fetal and adults red blood cells.

In human red blood cells from umbilical cord (NRBC) and from adult (ARBC) blood we measured: (i) the equilibrium distribution (medium/cells) for haemoglobin as a function of medium osmolality (osmotic fragility curve) and (ii) the rate of haemoglobin loss to a hypotonic medium of fixed osmolality. From the analysis of the osmotic (cumulative) fragility curve, a subpopulation of high resistance (young?) cells was individualized only in samples from cord blood. The differences presented in the rate of haemoglobin loss between samples of umbilical cord and adult's blood point to distinct cell surface restrictions (cytoskeleton and/or plasma membrane phospholipid) to haemoglobin leak in both cells. Uniformly distributed (among all cells in both samples) differences could explain the distinct rates of haemoglobin loss. However, marked differences restricted to only a subpopulation of cord blood cells could also explain these results.