Stimulation of human erythrocyte K-Cl cotransport and protein phosphatase type 2A by n-ethylmaleimide: role of intracellular Mg++.

An increase in the activity of membrane-associated protein phosphatase type 1 (mb-PP1) is associated with stimulation of erythrocyte K-Cl cotransport (KCC). We have recently proposed that membrane-associated protein phosphatase type 2A (mb-PP2A) is also involved in KCC regulation by cell swelling (Bize et al., 1999. Am. J. Physiol. 277:C899-C912). We used two protein phosphatase inhibitors, okadaic acid (OA) and calyculin A (CalA), and two KCC activating treatments, n-ethylmaleimide (NEM) and Mg(i)++-depletion, and determined KCC transport activity and mb-PP1 and mb-PP2A activities. OA, an inhibitor of erythrocyte mb-PP2A, partially prevents stimulation of KCC activity by NEM but not by Mg(i)++-depletion. CalA, an inhibitor of both mb-PP1 and mb-PP2A prevents stimulation of KCC activity by both treatments. NEM and Mg(i)++-depletion inhibit mb-PP1 activity, suggesting that activation of KCC can take place in the absence of mb-PP1 activation. Mb-PP2A activity is stimulated in NEM-treated cells but not in Mg(i)++-depleted cells. In NEM-treated cells, Mg(i)++-depletion inhibits both KCC and mb-PP2A. In Mg(i)++-depleted cells, NEM does not stimulate KCC or mb-PP2A. The strong correlation between KCC stimulation and mb-PP2A stimulation provides further support to the idea that mb-PP2A plays an important role in KCC regulation. Our results are consistent with the hypothesis that KCC regulation involves at least two distinguishable phosphorylation sites.

Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes.

Activation of K-Cl cotransport is associated with activation of membrane-bound serine/threonine protein phosphatases (S/T-PPases). We characterize red blood cell S/T-PPases and K-Cl cotransport activity regarding protein phosphatase inhibitors and response to changes in ionic strength and cell size. Protein phosphatase type 1 (PP1) activity is highly sensitive to calyculin A (CalA) but not to okadaic acid (OA). PP2A activity is highly sensitive to CalA and OA. CalA completely inhibits K-Cl cotransport activity, whereas OA partially inhibits K-Cl cotransport. Membrane PP1 and membrane PP2A activities are elevated in cells suspended in hypotonic solutions, where K-Cl cotransport is elevated. Increases in membrane PP1 activity (62 +/- 10% per 100 meq/l) result from decreases in intracellular ionic strength and correlate with increases in K-Cl cotransport activity (54 +/- 10% per 100 meq/l). Increases in membrane PP2A activity (270 +/- 77% per 100 mosM) result from volume increases and also correlate with increases in K-Cl cotransport activity (420 +/- 47% per 100 mosM). The characteristics of membrane-associated PP1 and PP2A are consistent with a role for both phosphatases in K-Cl cotransport activation in human erythrocytes.

Characterization of a model of malaria in the pregnant host: Plasmodium berghei in the white rat.

This study characterizes a Plasmodium berghei white rat model of P. falciparum malaria in the pregnant human. Seventy-day-old and 114-day-old female rats, given an infecting inoculum at time of mating, had higher parasitemias and a more severe anemia than age- and sex-matched controls. Under these experimental conditions, the parasitemia went to crisis in all animals and there were no fatal infections. In contrast, all animals died when the infection was initiated 7 days after conception, a timing that brought a coincidence of peak parasitemia and term. Pregnancy during the post-crisis subpatent period did not cause recrudescence. At the time of delivery, the parasitemia was consistently higher in the placental (crush smear) blood than in the peripheral (tail) blood. This difference was greatest in animals giving birth shortly before or 1-2 days after the parasitemic crisis. Very young, compact parasite forms predominated in the placental blood, whereas trophozoites predominated in the peripheral blood.