Patients and methods

Results

Table 1 shows the relative abundance of the Na⁺,K⁺-ATPase subunit isoforms studied in the three groups of neonates. We did not find any evidence for the presence of the α₃ subunit isoform in neonatal erythrocytes, but α₁, α₂, β₁ and β₂ subunit isoforms were readily detected. The abundance of the α₂ and β₁ subunit isoforms remained unchanged during the studied period of human development. However, the abundance of the α₁ isoforms decreased while that of the β₂ isoforms increased with advancing gestational age. The (α₁+α₂)/(β₁+β₂) ratio was 89% higher in the preterm neonates in group 1 than in the term neonates (group 3). However, as specific antisera to the α₄ and β₃ isoforms were not available at the time of the study, we cannot comment on the changes in the total α/β subunit ratio. Figure 1 shows a representative western blot developed by enhanced chemiluminescence illustrating the developmental regulation of erythrocyte Na⁺,K⁺-ATPase α₁ and β₂ subunit isoforms in nine characteristic samples.

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Figure 1

Immunoblot analysis of Na⁺,K⁺-ATPase α₁, α₂, β₁ and β₂ subunit isoforms of neonatal erythrocytes. A representative western blot developed by enhanced chemiluminescence is shown obtained from pooled blood collected from patients in group 1 (gestational age 28–32 weeks; lanes 1–3), group 2 (gestational age 33–36 weeks; lanes 4–6), and group 3 (gestational age 37–42 weeks; lanes 7–9). Molecular mass markers run on the same gel were phosphorylase b (112 kDa) and ovalbumin (53 kDa). See the text for details.

Table 2 gives the Na⁺,K⁺-ATPase activity of the enzyme and [Na⁺]_ic and [K⁺]_ic in the three groups of neonates. Erythrocyte cell membrane Na⁺,K⁺-ATPase activity decreased with advancing gestational age, and there was an inverse relation between Na⁺,K⁺-ATPase activity and gestational age (y = 1219−20.6x,r = 0.345, p = 0.0001; fig 2A) or birth weight (p = 0.0011; not shown). [Na⁺]_ic was significantly higher in group 3 than in groups 1 and 2 (table 2), and there was a significant direct relation between [Na⁺]_ic and gestational age (y = 2.72 + 0.136x, r = 0.342, p = 0.0025; fig 2B) or birth weight (p = 0.0048; not shown). There was no difference in [K⁺]_ic among the groups and no correlation could be found between [K⁺]_ic and gestational age and birth weight (p = 0.143 and 0.387 respectively; simple regression analysis, not shown).

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Figure 2

Relation of gestational age to purified erythrocyte membrane Na⁺,K⁺-ATPase activity (A) and intracellular Na⁺ concentration (B). Erythrocyte membrane Na⁺,K⁺-ATPase activity is inversely related to gestational age (y = 1218.7−20.6x, r = 0.345; p = 0.0001; simple regression analysis), and intracellular Na⁺concentration (mmol/l erythrocyte volume) relates directly to gestational age (y = 2.715 + 0.136x, r = 0.342; p = 0.0025; simple regression analysis). See the text for details.

Discussion

This study shows that erythrocyte Na⁺,K⁺-ATPase α₁ and β₂ subunit isoform expression is developmentally regulated in the neonate. We found a maturation dependent decrease in α₁ and an increase in β₂ subunit isoform abundance in neonates between 28 and 42 weeks of gestation. Our results also show that α₂ and β₁ subunit isoform abundance is not affected by maturation and that neonatal erythrocytes do not possess α₃ subunit isoforms. As the α₁ and β₁ subunit isoforms are the most commonly expressed isoforms in most of the tissues outside the nervous system,2 ,5 Na⁺,K⁺-ATPase activity and cation pump function are thought to be primarily determined by the abundance of these isoforms. In agreement with this notion, erythrocyte Na⁺,K⁺-ATPase activity was found to decrease with maturation, correlating with the changes in α₁subunit abundance. Finally, the higher [Na⁺]_ic of mature neonates may represent a decrease in their erythrocyte Na⁺,K⁺-ATPase cation pump function commensurate with the developmentally regulated changes in ATPase activity. However, as unidirectional Na⁺fluxes were not studied, developmentally regulated changes in the expression and/or function of Na⁺ leak pathways may have contributed to this finding.

Only limited information is available on the developmental regulation of erythrocyte Na⁺,K⁺-ATPase subunit isoform expression in man. We have recently shown that erythrocytes of term neonates express more α₁ subunit isoforms and have higher erythrocyte membrane Na⁺,K⁺-ATPase activity than those of children.15 In the present study, we extend this information to earlier stages of human development and provide convincing evidence that erythrocyte α₁ subunit isoform expression is developmentally regulated (table 1, fig 1). It is tempting to speculate that the upregulation of α₁ subunit expression during early development is, at least in part, related to the developmentally regulated high levels of endogenous inhibitors of Na⁺,K⁺-ATPase in the immature neonate.17 ,18 The fact that the Na⁺,K⁺-ATPase enzyme possessing the α₁ isoform is the most resistant to these endogenous digoxin-like substances1 ,2 ,5 makes this hypothesis even more attractive and suggests that there is a compensatory mechanism to maintain Na⁺,K⁺-ATPase function in the presence of higher concentrations of endogenous inhibitors.12

In agreement with our findings, most previous studies have also found higher erythrocyte Na⁺,K⁺-ATPase activity6 ,10 ,11 and ouabain binding capacity12 in the immature animal and neonate than in the mature newborn. On the other hand, contrary to our present and the above mentioned previous findings,6 ,10-12 a recent study by Bistritzer et al 13 found that cord blood erythrocyte ATPase activity is lower in preterm neonates of between 30 and 34 weeks of gestation than neonates of ⩾ 35 weeks gestation. However, as Na⁺,K⁺-ATPase activity has been reported to increase after birth in preterm neonates,8 it is possible that the use of cord blood by Bistritzer et al affected their findings. In support of this hypothesis is the fact that they themselves reported a postnatal increase in erythrocyte Na⁺,K⁺-ATPase activity in their preterm neonates.13 Finally, our findings are strengthened by the enrollment of a larger patient population including more immature preterm neonates and by the downregulation of the α₁subunit isoform in term neonates, suggesting the presence of an indirect relation between erythrocyte Na⁺,K⁺-ATPase activity and gestational age.

We found lower [Na⁺]_ic in preterm neonates than in term neonates (table 2). In addition, there is also a direct relation between [Na⁺]_ic and gestational age (fig 2B) or birth weight. In agreement with these findings, other investigators have also reported a positive correlation between erythrocyte [Na⁺]_ic and gestational age.6 ,8 ,11 ,12 Thus it is tempting to speculate that, in addition to their increased ATPase activity, immature neonates also have a higher erythrocyte Na⁺,K⁺-ATPase cation pump function. As for the [K⁺]_ic, previous studies8 ,11 have also found that [K⁺]_ic does not necessarily change with maturation (table 2). However, because of the high [K⁺]_ic and the unique stoichiometry of the enzyme, it is generally believed that [K⁺]_icis a less sensitive marker of Na⁺,K⁺-ATPase cation pump function than [Na⁺]_ic.10 Finally, as mentioned above, developmentally regulated changes in the expression and/or function of the Na⁺ and K⁺ leak pathways may have contributed to the changes in intracellular cation concentrations.

In summary, of the five erythrocyte Na⁺,K⁺-ATPase subunit isoforms studied, only the α₁ and β₂ isoforms are developmentally regulated. The increased α₁ isoform abundance of preterm neonates is associated with enhanced erythrocyte Na⁺,K⁺-ATPase activity and, on the basis of the findings for [Na⁺]_ic, with an increased cation pump function of the enzyme. Finally, it is important to emphasise that the findings in erythrocytes may not represent the relation between Na⁺,K⁺-ATPase subunit isoform composition and enzyme function in other tissues. Therefore caution should be exercised when speculating about the direct clinical significance of these findings.