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Am J Physiol Cell Physiol (February 26, 2003). doi:10.1152/ajpcell.00447.2002
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Submitted on September 27, 2002
Accepted on February 22, 2003

Regulation of K-Cl cotransport during reticulocyte maturation and erythrocyte aging in normal and sickle erythrocytes

Isabel Bize1, Samara Taher2, and Carlo Brugnara2*

1 Departments of Cell Biology, Harvard Medical School, Booston, MA, USA
2 Department of Laboratory Medicine, Children's Hospital Boston, Boston, MA, USA

* To whom correspondence should be addressed. E-mail: carlo.brugnara{at}tch.harvard.edu.

The age/density-dependent decrease in K-Cl cotransport (KCC), PP1 and PP2A activities in normal and sickle human erythrocytes and the effect of urea, a known KCC activator, were studied using discontinuous, isotonic gradients. In normal erythrocytes, the densest fraction (d ~33.4 g/dl) has only about ~5% of the KCC and 4% of the membrane (mb)-PP1 activities of the least-dense fraction (d ~24.7 g/dl). In sickle and normal erythrocytes, density-dependent decreases for mb-PP1 activity were similar (d50% 28.1 ± 0.4 g/dl vs. 27.2 ± 0.2 g/dl, respectively) while those KCC activity were not (d50% 31.4 ± 0.9 g/dl vs. 26.8 ± 0.3 g/dl, , respectively, p = 0.004). Excluding the 10% least-dense cells, a very tight correlation exists between KCC and mb-PP1 activities in normal (r2 = 0.995), and sickle erythrocytes (r2=0.93), but at comparable mb-PP1 activities, KCC activity in higher in sickle erythrocytes, suggesting a defective, mb-PP1 independent, KCC regulation. In normal, least-dense but not in densest cells, urea stimulates KCC (2-4 fold) and moderately increases mb-PP1 (20-40%). Thus, mb-PP1 appears to mediate part of urea-stimulated KCC activity.




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