Lead ions but not other metallic ions increase resistance to hypotonic lysis in prenatal hemopoiesis red blood cells

Metals known to have toxic effects on exposed individuals (Aluminum (Al), Cadmium (Cd), Zinc (Zn) and lead (Pb)) were selected. Umbilical cord erythrocytes from normal newborns were incubated in isotonic media alone or with addition of Pb (20 microM), Cd, Zn or Al (concentration range: 20-250 microM). Red cells were then placed in media of diminishing tonicity, to measure cellular lysis and volume; the regression curves of percent lysis as a function of osmolarity were determined for each data set and the break points calculated. Resistance to lysis increased significantly in Pb treated cells whereas cells treated with the other metals did not differ from controls, even at concentrations ten times higher than that of Pb. Lead produced a reduction in cellular volume corrected by addition of quinidine (an inhibitor of potassium channels activation) to the cell suspension; on the other hand, quinidine did not modify the effect of lead on lysis sensitivity. These results suggest that the effect of lead on cell resistance to lysis might be mediated by changes in membrane structure. The other metals examined did not affect the variables studied.(AU)

Lead ions but not other metallic ions increase resistance to hypotonic lysis in prenatal hemopoiesis red blood cells

Metals known to have toxic effects on exposed individuals (Aluminum (Al), Cadmium (Cd), Zinc (Zn) and lead (Pb)) were selected. Umbilical cord erythrocytes from normal newborns were incubated in isotonic media alone or with addition of Pb (20 microM), Cd, Zn or Al (concentration range: 20-250 microM). Red cells were then placed in media of diminishing tonicity, to measure cellular lysis and volume; the regression curves of percent lysis as a function of osmolarity were determined for each data set and the break points calculated. Resistance to lysis increased significantly in Pb treated cells whereas cells treated with the other metals did not differ from controls, even at concentrations ten times higher than that of Pb. Lead produced a reduction in cellular volume corrected by addition of quinidine (an inhibitor of potassium channels activation) to the cell suspension; on the other hand, quinidine did not modify the effect of lead on lysis sensitivity. These results suggest that the effect of lead on cell resistance to lysis might be mediated by changes in membrane structure. The other metals examined did not affect the variables studied.

Lead ions but not other metallic ions increase resistance to hypotonic lysis in prenatal hemopoiesis red blood cells.

Metals known to have toxic effects on exposed individuals (Aluminum (Al), Cadmium (Cd), Zinc (Zn) and lead (Pb)) were selected. Umbilical cord erythrocytes from normal newborns were incubated in isotonic media alone or with addition of Pb (20 microM), Cd, Zn or Al (concentration range: 20-250 microM). Red cells were then placed in media of diminishing tonicity, to measure cellular lysis and volume; the regression curves of percent lysis as a function of osmolarity were determined for each data set and the break points calculated. Resistance to lysis increased significantly in Pb treated cells whereas cells treated with the other metals did not differ from controls, even at concentrations ten times higher than that of Pb. Lead produced a reduction in cellular volume corrected by addition of quinidine (an inhibitor of potassium channels activation) to the cell suspension; on the other hand, quinidine did not modify the effect of lead on lysis sensitivity. These results suggest that the effect of lead on cell resistance to lysis might be mediated by changes in membrane structure. The other metals examined did not affect the variables studied.

Lead ions but not other metallic ions increase resistance to hypotonic lysis in prenatal hemopoiesis red blood cells.

Metals known to have toxic effects on exposed individuals (Aluminum (Al), Cadmium (Cd), Zinc (Zn) and lead (Pb)) were selected. Umbilical cord erythrocytes from normal newborns were incubated in isotonic media alone or with addition of Pb (20 microM), Cd, Zn or Al (concentration range: 20-250 microM). Red cells were then placed in media of diminishing tonicity, to measure cellular lysis and volume; the regression curves of percent lysis as a function of osmolarity were determined for each data set and the break points calculated. Resistance to lysis increased significantly in Pb treated cells whereas cells treated with the other metals did not differ from controls, even at concentrations ten times higher than that of Pb. Lead produced a reduction in cellular volume corrected by addition of quinidine (an inhibitor of potassium channels activation) to the cell suspension; on the other hand, quinidine did not modify the effect of lead on lysis sensitivity. These results suggest that the effect of lead on cell resistance to lysis might be mediated by changes in membrane structure. The other metals examined did not affect the variables studied.

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.

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 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.